GcatWiki - User contributions [en]

Logic Gates - Emma Garren

2007-12-14T02:33:41Z

Emgarren: /* Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro ''et al.'', 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro ''et al.'', 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer ''et al.'', 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary: Many eukaryotic signaling proteins have natural, modular "input" and "output" domains: the "inputs" participate in steric or conformational autoinhibitory reactions, and the "outputs" are catalytically, constitutively active domains. Simple synthetic switch functions can be engineered by swapping the regulatory and catalytic domains. This paper describes the engineering of "ultrasensitive switches" for use in more complex regulatory networks, by combining multiple identical modular autoinhibitory domains that function [http://en.wikipedia.org/wiki/Cooperativity cooperatively]. Mathematical models are used to simulate the behavior of these multivalent domain switches and explore the effect of autoinhibitory interaction number and affinity: an external input ligand alters the population distribution of active vs. inactive enzymes, where individual states are "fully repressed in the presence of any intramolecular interactions and fully active only in the absence of all intramolecular interactions" (661). The predictions from the models are tested using synthetic switches built by linking the catalytic output domain of the protein N-WASP to novel peptide input, demonstrating that it is possible to engineer nonlinear switches based on the cooperativity of simple autoinhibitory components. Other complex switches, such as ones that integrate three input signals, have also been built using this approach.

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

===Overview:===

This gate uses sequence recognition to control enzyme catalysis of covalent bond formation and breakage, producing an ssDNA output.

*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

===Design:===

[[Figure 2. Logical design of the molecular computer.]]

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

===Function:===

[[Figure 3. Operation of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

The benefits of this approach are that both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed of one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

As stated above, there the cellular environment possesses many characteristics that complicate the implementation of biomolecular logic circuits, such as gene expression noise, mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context. Some researchers are trying to engineer the "minimal cell," either through top-down or bottom-up approaches, in order to produce a living unit that can perform logical operations without some of these excess confounding factors. These efforts were recently reviewed by [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf Forster and Church (2006)]. However, many efforts at building cellular logic gates have succeeded, both in prokaryotes and eukaryotes, with wide-ranging applications. Below are summaries of a few examples.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed in ''E. coli'' based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering:
**increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]:
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Forster, A.C., Church G.M. (2006). Towards synthesis of a minimal cell. Mol Sys Biol. 2(45):1-10. [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf PDF]
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-14T02:32:39Z

Emgarren: /* Cellular Logic Gates: ''In Vivo'' */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro ''et al.'', 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro ''et al.'', 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer ''et al.'', 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary: Many eukaryotic signaling proteins have natural, modular "input" and "output" domains: the "inputs" participate in steric or conformational autoinhibitory reactions, and the "outputs" are catalytically, constitutively active domains. Simple synthetic switch functions can be engineered by swapping the regulatory and catalytic domains. This paper describes the engineering of "ultrasensitive switches" for use in more complex regulatory networks, by combining multiple identical modular autoinhibitory domains that function [http://en.wikipedia.org/wiki/Cooperativity cooperatively]. Mathematical models are used to simulate the behavior of these multivalent domain switches and explore the effect of autoinhibitory interaction number and affinity: an external input ligand alters the population distribution of active vs. inactive enzymes, where individual states are "fully repressed in the presence of any intramolecular interactions and fully active only in the absence of all intramolecular interactions" (661). The predictions from the models are tested using synthetic switches built by linking the catalytic output domain of the protein N-WASP to novel peptide input, demonstrating that it is possible to engineer nonlinear switches based on the cooperativity of simple autoinhibitory components. Other complex switches, such as ones that integrate three input signals, have also been built using this approach.

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

===Overview:===

This gate uses sequence recognition to control enzyme catalysis of covalent bond formation and breakage, producing an ssDNA output.

*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

===Design:===

[[Figure 2. Logical design of the molecular computer.]]

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

===Function:===

[[Figure 3. Operation of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

The benefits of this approach are that both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed of one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

As stated above, there the cellular environment possesses many characteristics that complicate the implementation of biomolecular logic circuits, such as gene expression noise, mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context. Some researchers are trying to engineer the "minimal cell," either through top-down or bottom-up approaches, in order to produce a living unit that can perform logical operations without some of these excess confounding factors. These efforts were recently reviewed by [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf Forster and Church (2006)]. However, many efforts at building cellular logic gates have succeeded, both in prokaryotes and eukaryotes, with wide-ranging applications. Below are summaries of a few examples.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering:
**increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]:
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Forster, A.C., Church G.M. (2006). Towards synthesis of a minimal cell. Mol Sys Biol. 2(45):1-10. [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf PDF]
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T17:21:20Z

Emgarren: /* Applications and Future Directions */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary: Many eukaryotic signaling proteins have natural, modular "input" and "output" domains: the "inputs" participate in steric or conformational autoinhibitory reactions, and the "outputs" are catalytically, constitutively active domains. Simple synthetic switch functions can be engineered by swapping the regulatory and catalytic domains. This paper describes the engineering of "ultrasensitive switches" for use in more complex regulatory networks, by combining multiple identical modular autoinhibitory domains that function [http://en.wikipedia.org/wiki/Cooperativity cooperatively]. Mathematical models are used to simulate the behavior of these multivalent domain switches and explore the effect of autoinhibitory interaction number and affinity: an external input ligand alters the population distribution of active vs. inactive enzymes, where individual states are "fully repressed in the presence of any intramolecular interactions and fully active only in the absence of all intramolecular interactions" (661). The predictions from the models are tested using synthetic switches built by linking the catalytic output domain of the protein N-WASP to novel peptide input, demonstrating that it is possible to engineer nonlinear switches based on the cooperativity of simple autoinhibitory components. Other complex switches, such as ones that integrate three input signals, have also been built using this approach.

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

===Overview:===

This gate uses sequence recognition to control enzyme catalysis of covalent bond formation and breakage, producing an ssDNA output.

*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

===Design:===

[[Figure 2. Logical design of the molecular computer.]]

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

===Function:===

[[Figure 3. Operation of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

The benefits of this approach are that both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed of one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

As stated above, there the cellular environment possesses many characteristics that complicate the implementation of biomolecular logic circuits, such as gene expression noise, mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context. Some researchers are trying to engineer the "minimal cell," either through top-down or bottom-up approaches, in order to produce a living unit that can perform logical operations without some of these excess confounding factors found in the cells used . These efforts were recently reviewed by [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf Forster and Church (2006)]. However, many efforts at building cellular logic gates have succeeded, both in prokaryotes and eukaryotes, with wide-ranging applications. Below are summaries of a few examples.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering:
**increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]:
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Forster, A.C., Church G.M. (2006). Towards synthesis of a minimal cell. Mol Sys Biol. 2(45):1-10. [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf PDF]
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T17:20:11Z

Emgarren: /* Synthetic Oscillators and Switches */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary: Many eukaryotic signaling proteins have natural, modular "input" and "output" domains: the "inputs" participate in steric or conformational autoinhibitory reactions, and the "outputs" are catalytically, constitutively active domains. Simple synthetic switch functions can be engineered by swapping the regulatory and catalytic domains. This paper describes the engineering of "ultrasensitive switches" for use in more complex regulatory networks, by combining multiple identical modular autoinhibitory domains that function [http://en.wikipedia.org/wiki/Cooperativity cooperatively]. Mathematical models are used to simulate the behavior of these multivalent domain switches and explore the effect of autoinhibitory interaction number and affinity: an external input ligand alters the population distribution of active vs. inactive enzymes, where individual states are "fully repressed in the presence of any intramolecular interactions and fully active only in the absence of all intramolecular interactions" (661). The predictions from the models are tested using synthetic switches built by linking the catalytic output domain of the protein N-WASP to novel peptide input, demonstrating that it is possible to engineer nonlinear switches based on the cooperativity of simple autoinhibitory components. Other complex switches, such as ones that integrate three input signals, have also been built using this approach.

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

===Overview:===

This gate uses sequence recognition to control enzyme catalysis of covalent bond formation and breakage, producing an ssDNA output.

*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

===Design:===

[[Figure 2. Logical design of the molecular computer.]]

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

===Function:===

[[Figure 3. Operation of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

The benefits of this approach are that both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed of one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

As stated above, there the cellular environment possesses many characteristics that complicate the implementation of biomolecular logic circuits, such as gene expression noise, mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context. Some researchers are trying to engineer the "minimal cell," either through top-down or bottom-up approaches, in order to produce a living unit that can perform logical operations without some of these excess confounding factors found in the cells used . These efforts were recently reviewed by [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf Forster and Church (2006)]. However, many efforts at building cellular logic gates have succeeded, both in prokaryotes and eukaryotes, with wide-ranging applications. Below are summaries of a few examples.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Forster, A.C., Church G.M. (2006). Towards synthesis of a minimal cell. Mol Sys Biol. 2(45):1-10. [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf PDF]
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T17:19:23Z

Emgarren: /* Protein-based Logic Gates */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary: Many eukaryotic signaling proteins have natural, modular "input" and "output" domains: the "inputs" participate in steric or conformational autoinhibitory reactions, and the "outputs" are catalytically, constitutively active domains. Simple synthetic switch functions can be engineered by swapping the regulatory and catalytic domains. This paper describes the engineering of "ultrasensitive switches" for use in more complex regulatory networks, by combining multiple identical modular autoinhibitory domains that function [http://en.wikipedia.org/wiki/Cooperativity cooperatively]. Mathematical models are used to simulate the behavior of these multivalent domain switches and explore the effect of autoinhibitory interaction number and affinity: an external input ligand alters the population distribution of active vs. inactive enzymes, where individual states are "fully repressed in the presence of any intramolecular interactions and fully active only in the absence of all intramolecular interactions" (661). The predictions from the models are tested using synthetic switches built by linking the catalytic output domain of the protein N-WASP to novel peptide input, demonstrating that it is possible to engineer nonlinear switches based on the cooperativity of simple autoinhibitory components. Other complex switches, such as ones that integrate three input signals, have also been built using this approach.

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

===Overview:===

This gate uses sequence recognition to control enzyme catalysis of covalent bond formation and breakage, producing an ssDNA output.

*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

===Design:===

[[Figure 2. Logical design of the molecular computer.]]

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

===Function:===

[[Figure 3. Operation of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

The benefits of this approach are that both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed of one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

As stated above, there the cellular environment possesses many characteristics that complicate the implementation of biomolecular logic circuits, such as gene expression noise, mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context. Some researchers are trying to engineer the "minimal cell," either through top-down or bottom-up approaches, in order to produce a living unit that can perform logical operations without some of these excess confounding factors found in the cells used . These efforts were recently reviewed by [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf Forster and Church (2006)]. However, many efforts at building cellular logic gates have succeeded, both in prokaryotes and eukaryotes, with wide-ranging applications. Below are summaries of a few examples.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]
(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Forster, A.C., Church G.M. (2006). Towards synthesis of a minimal cell. Mol Sys Biol. 2(45):1-10. [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf PDF]
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T17:12:24Z

Emgarren: /* Protein-based Logic Gates */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary: Many eukaryotic signaling proteins have natural, modular "input" and "output" domains: the "inputs" participate in steric or conformational autoinhibitory reactions, and the "outputs" are catalytically, constitutively active domains. Simple synthetic switch functions can be engineered by swapping the regulatory and catalytic domains. This paper describes the engineering of "ultrasensitive switches" for use in more complex regulatory networks, by combining multiple identical modular autoinhibitory domains that function [http://en.wikipedia.org/wiki/Cooperativity cooperatively]. The authors used mathematical models to simulate the behavior of these multivalent domain switches and explore the effect of autoinhibitory interaction number and affinity: an external input ligand alters the population distribution of active vs. inactive enzymes, where individual states are "fully repressed in the presence of any intramolecular interactions and fully active only in the absence of all intramolecular interactions" (661).

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

===Overview:===

This gate uses sequence recognition to control enzyme catalysis of covalent bond formation and breakage, producing an ssDNA output.

*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

===Design:===

[[Figure 2. Logical design of the molecular computer.]]

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

===Function:===

[[Figure 3. Operation of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

The benefits of this approach are that both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed of one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

As stated above, there the cellular environment possesses many characteristics that complicate the implementation of biomolecular logic circuits, such as gene expression noise, mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context. Some researchers are trying to engineer the "minimal cell," either through top-down or bottom-up approaches, in order to produce a living unit that can perform logical operations without some of these excess confounding factors found in the cells used . These efforts were recently reviewed by [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf Forster and Church (2006)]. However, many efforts at building cellular logic gates have succeeded, both in prokaryotes and eukaryotes, with wide-ranging applications. Below are summaries of a few examples.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]
(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Forster, A.C., Church G.M. (2006). Towards synthesis of a minimal cell. Mol Sys Biol. 2(45):1-10. [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf PDF]
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T17:12:08Z

Emgarren: /* Protein-based Logic Gates */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary: Many eukaryotic signaling proteins have natural, modular "input" and "output" domains: the "inputs" participate in steric or conformational autoinhibitory reactions, and the "outputs" are catalytically, constitutively active domains. Simple synthetic switch functions can be engineered by swapping the regulatory and catalytic domains. This paper describes the engineering of "ultrasensitive switches" for use in more complex regulatory networks, by combining multiple identical modular autoinhibitory domains that function [[http://en.wikipedia.org/wiki/Cooperativity cooperatively]]. The authors used mathematical models to simulate the behavior of these multivalent domain switches and explore the effect of autoinhibitory interaction number and affinity: an external input ligand alters the population distribution of active vs. inactive enzymes, where individual states are "fully repressed in the presence of any intramolecular interactions and fully active only in the absence of all intramolecular interactions" (661).

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

===Overview:===

This gate uses sequence recognition to control enzyme catalysis of covalent bond formation and breakage, producing an ssDNA output.

*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

===Design:===

[[Figure 2. Logical design of the molecular computer.]]

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

===Function:===

[[Figure 3. Operation of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

The benefits of this approach are that both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed of one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

As stated above, there the cellular environment possesses many characteristics that complicate the implementation of biomolecular logic circuits, such as gene expression noise, mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context. Some researchers are trying to engineer the "minimal cell," either through top-down or bottom-up approaches, in order to produce a living unit that can perform logical operations without some of these excess confounding factors found in the cells used . These efforts were recently reviewed by [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf Forster and Church (2006)]. However, many efforts at building cellular logic gates have succeeded, both in prokaryotes and eukaryotes, with wide-ranging applications. Below are summaries of a few examples.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]
(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Forster, A.C., Church G.M. (2006). Towards synthesis of a minimal cell. Mol Sys Biol. 2(45):1-10. [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf PDF]
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

File:Benenson sup figure.jpg

2007-12-06T06:16:10Z

Emgarren:

Restoration: Thresholding Gate

2007-12-06T06:10:06Z

Emgarren:

A signal restoration module consists of a threshold gate and an amplification gate, and its "incorporation into large circuits at multiple intermediate points ensures digital representation" (1587):

#Threshold gate: The threshold gate consists of three-input AND gate with identical first and third inputs. The second input is only necessary for structural purposes, and the third input is hybridized to an output strand. An insufficient amount of input will cause most gates to lose only their first and second gate strands, and not the third, therefore failing to release output.
#Amplification gate: An amplification gate is necessary because "the threshold gate's output cannot exceed half the input signal" (1587), and therefore must be amplified. Two methods for building an amplification gate are described: 1. A hybridization-based system for catalytic amplification, serving as both input amplifier and full translator, and 2. An amplifier based on feedback logic.

Restoration: Amplification Gate

2007-12-06T06:09:31Z

Emgarren:

Restoration: Amplification Gate

2007-12-06T06:09:07Z

Emgarren:

A signal restoration module consists of a threshold gate and an amplification gate, and its "incorporation into large circuits at multiple intermediate points ensures digital representation" (1587):

#Threshold gate: The threshold gate consists of three-input AND gate with identical first and third inputs. The second input is only necessary for structural purposes, and the third input is hybridized to an output strand. An insufficient amount of input will cause most gates to lose only their first and second gate strands, and not the third, therefore failing to release output.

#Amplification gate: An amplification gate is necessary because "the threshold gate's output cannot exceed half the input signal" (1587), and therefore must be amplified. Two methods for building an amplification gate are described: 1. A hybridization-based system for catalytic amplification, serving as both input amplifier and full translator, and 2. An amplifier based on feedback logic.

Restoration: Thresholding Gate

2007-12-06T06:08:36Z

Emgarren:

Logic Gates - Emma Garren

2007-12-06T05:55:08Z

Emgarren: /* Cellular Logic Gates: ''In Vivo'' */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

===Overview:===

This gate uses sequence recognition to control enzyme catalysis of covalent bond formation and breakage, producing an ssDNA output.

*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

===Design:===

[[Figure 2. Logical design of the molecular computer.]]

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

===Function:===

[[Figure 3. Operation of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

The benefits of this approach are that both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed of one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

As stated above, there the cellular environment possesses many characteristics that complicate the implementation of biomolecular logic circuits, such as gene expression noise, mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context. Some researchers are trying to engineer the "minimal cell," either through top-down or bottom-up approaches, in order to produce a living unit that can perform logical operations without some of these excess confounding factors found in the cells used . These efforts were recently reviewed by [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf Forster and Church (2006)]. However, many efforts at building cellular logic gates have succeeded, both in prokaryotes and eukaryotes, with wide-ranging applications. Below are summaries of a few examples.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]
(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Forster, A.C., Church G.M. (2006). Towards synthesis of a minimal cell. Mol Sys Biol. 2(45):1-10. [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf PDF]
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T05:51:39Z

Emgarren: /* Cellular Logic Gates: ''In Vivo'' */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

===Overview:===

This gate uses sequence recognition to control enzyme catalysis of covalent bond formation and breakage, producing an ssDNA output.

*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

===Design:===

[[Figure 2. Logical design of the molecular computer.]]

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

===Function:===

[[Figure 3. Operation of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

The benefits of this approach are that both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed of one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

As stated above, there the cellular environment possesses many characteristics that complicate the implementation of biomolecular logic circuits, such as gene expression noise, mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context. Some researchers are trying to engineer the "minimal cell," either through top-down or bottom-up approaches, in order to produce something that can perform the logical operations without some of these excess confounding factors. These efforts were recently reviewed by [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf Forster and Church (2006)]. However, many efforts at building cellular logic gates have succeeded, both in prokaryotes and eukaryotes, with wide-ranging applications. Below is a summary of a few examples.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]
(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Forster, A.C., Church G.M. (2006). Towards synthesis of a minimal cell. Mol Sys Biol. 2(45):1-10. [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf PDF]
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T05:49:35Z

Emgarren: /* References */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

===Overview:===

This gate uses sequence recognition to control enzyme catalysis of covalent bond formation and breakage, producing an ssDNA output.

*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

===Design:===

[[Figure 2. Logical design of the molecular computer.]]

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

===Function:===

[[Figure 3. Operation of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

The benefits of this approach are that both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed of one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

As stated above, there the cellular environment possesses many characteristics that complicate the implementation of biomolecular logic circuits, such as gene expression noise, mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context. Some researchers are trying to engineer the "minimal cell," either through top-down or bottom-up approaches, in order to produce something that can perform the logical operations without some of these excess confounding factors. However, many efforts at building cellular logic gates have succeeded, both in prokaryotes and eukaryotes, and these have wide-ranging applications.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]
(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Forster, A.C., Church G.M. (2006). Towards synthesis of a minimal cell. Mol Sys Biol. 2(45):1-10. [http://www.nature.com/msb/journal/v2/n1/pdf/msb4100090.pdf PDF]
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T05:45:51Z

Emgarren: /* Cellular Logic Gates: ''In Vivo'' */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

===Overview:===

This gate uses sequence recognition to control enzyme catalysis of covalent bond formation and breakage, producing an ssDNA output.

*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

===Design:===

[[Figure 2. Logical design of the molecular computer.]]

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

===Function:===

[[Figure 3. Operation of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

The benefits of this approach are that both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed of one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

As stated above, there the cellular environment possesses many characteristics that complicate the implementation of biomolecular logic circuits, such as gene expression noise, mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context. Some researchers are trying to engineer the "minimal cell," either through top-down or bottom-up approaches, in order to produce something that can perform the logical operations without some of these excess confounding factors. However, many efforts at building cellular logic gates have succeeded, both in prokaryotes and eukaryotes, and these have wide-ranging applications.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]
(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T05:38:08Z

Emgarren: /* Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

===Overview:===

This gate uses sequence recognition to control enzyme catalysis of covalent bond formation and breakage, producing an ssDNA output.

*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

===Design:===

[[Figure 2. Logical design of the molecular computer.]]

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

===Function:===

[[Figure 3. Operation of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

The benefits of this approach are that both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed of one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]
(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Figure 3. Operation of the molecular computer.

2007-12-06T05:32:52Z

Emgarren:

"A novel molecular mechanism... regulates the probability of each positive transition by the corresponding molecular indicator, so that the presence of the indicator increases the probability of a positive transition and decreases the probability of its competing negative transition, and vice versa if the indicator is absent" (p. 424).

[[Image:Benenson_fig_2.gif]]

(Image from Benenson et al., 2004. Permission pending.)

Logic Gates - Emma Garren

2007-12-06T05:32:36Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

===Overview:===

This gate uses sequence recognition to control enzyme catalysis of covalent bond formation and breakage, producing an ssDNA output.

*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

===Design:===

[[Figure 2. Logical design of the molecular computer.]]

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

===Function:===

[[Figure 3. Operation of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]
(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T05:23:11Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

Overview:

This gate uses sequence recognition to control enzyme catalysis of covalent bond formation and breakage, producing an ssDNA output.

*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 2. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 3. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]
(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T05:19:31Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

Overview:

This gate uses sequence recognition to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 2. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 3. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]
(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T05:18:27Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 2. Logical design of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 2. Logical design of the molecular computer.]]

[[Image:Benenson_fig_2.gif|thumb|[[Figure 3. Operation of the molecular computer]]]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 3. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]
(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T05:18:17Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 2. Logical design of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 2. Logical design of the molecular computer.]]

[[Image:Benenson_fig_2.jpg|thumb|[[Figure 3. Operation of the molecular computer]]]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 3. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]
(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T05:17:50Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 2. Logical design of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 2. Logical design of the molecular computer.]]

[[Image:Benenson_fig_2.gif|thumb|[[Figure 3. Operation of the molecular computer]]]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 3. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]
(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T05:17:04Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 2. Logical design of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 2. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 3. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]
(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T04:01:16Z

Emgarren: /* Synthetic Oscillators and Switches */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 2. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 3. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]
(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

Figure 5. Schematic representation of the function of various engineered biological switches.

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T03:58:21Z

Emgarren: /* Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 2. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 3. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 4. Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Figure 4. Two-input AND gate.

2007-12-06T03:57:33Z

Emgarren: Figure 1: Two-input AND gate. moved to Figure 4. Two-input AND gate.

Figure 1. Two-Input AND gate.

The gate is composed of two gate strands and one output strand, and is initially double-stranded, and therefore inert, with the exception of the "toehold." The computation is initiated by the addition of single-stranded inputs. The first input binds to the toehold, displacing the first gate strand and exposing the toehold for the second input. The second input binds the toehold, releasing the output strand.

If one of the gate strands contains a fluorophore, and the output strand contains a quencher, the release of the output strand will result in an increase in fluorescence.

[[Image:Enzyme-free_fig_1.gif]]

(Image from Seelig et al., 2006. Permission pending.)

Figure 1: Two-input AND gate.

2007-12-06T03:57:33Z

Emgarren: Figure 1: Two-input AND gate. moved to Figure 4. Two-input AND gate.

#redirect [[Figure 4. Two-input AND gate.]]

Logic Gates - Emma Garren

2007-12-06T03:57:10Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 2. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 3. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 1: Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Figure 3. Operation of the molecular computer.

2007-12-06T03:56:27Z

Emgarren: Figure 2. Operation of the molecular computer. moved to Figure 3. Operation of the molecular computer.

[[Image:Benenson_fig_2.gif]]

(Image from Benenson et al., 2004. Permission pending.)

Figure 2. Operation of the molecular computer.

2007-12-06T03:56:27Z

Emgarren: Figure 2. Operation of the molecular computer. moved to Figure 3. Operation of the molecular computer.

#redirect [[Figure 3. Operation of the molecular computer.]]

Logic Gates - Emma Garren

2007-12-06T03:56:14Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 2. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 2. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 1: Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T03:55:46Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 1. Logical design of the molecular computer.]]]]

[[Image:Benenson fig 2.gif|thumb|[[Figure 2. Operation of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 2. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 2. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 1: Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Figure 2. Logical design of the molecular computer.

2007-12-06T03:55:15Z

Emgarren: Figure 1. Logical design of the molecular computer. moved to Figure 2. Logical design of the molecular computer.

[[Image:Benenson_fig_1_small.jpg]]

(Image from Benenson et al., 2004. Permission pending.)

Figure 1. Logical design of the molecular computer.

2007-12-06T03:55:15Z

Emgarren: Figure 1. Logical design of the molecular computer. moved to Figure 2. Logical design of the molecular computer.

#redirect [[Figure 2. Logical design of the molecular computer.]]

Logic Gates - Emma Garren

2007-12-06T03:52:46Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 1. Logical design of the molecular computer.]]]]

[[Image:Benenson fig 2.gif|thumb|[[Figure 2. Operation of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 1. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 2. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 1: Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T03:51:52Z

Emgarren: /* Background: Logic Gates and Truth Tables */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Combination of logic gates: Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 1. Logical design of the molecular computer.]]]]

[[Image:Benenson_fig_2.gif|thumb|[[Figure 2. Operation of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 1. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 2. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 1: Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T03:51:21Z

Emgarren: /* Background: Logic Gates and Truth Tables */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 1. Logical design of the molecular computer.]]]]

[[Image:Benenson_fig_2.gif|thumb|[[Figure 2. Operation of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 1. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 2. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 1: Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T03:51:07Z

Emgarren: /* Background: Logic Gates and Truth Tables */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

FIGURE 1. Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 1. Logical design of the molecular computer.]]]]

[[Image:Benenson_fig_2.gif|thumb|[[Figure 2. Operation of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 1. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 2. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 1: Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T03:50:47Z

Emgarren: /* Background: Logic Gates and Truth Tables */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Figure 1. Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 1. Logical design of the molecular computer.]]]]

[[Image:Benenson_fig_2.gif|thumb|[[Figure 2. Operation of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 1. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 2. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 1: Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T03:50:14Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 1. Logical design of the molecular computer.]]]]

[[Image:Benenson_fig_2.gif|thumb|[[Figure 2. Operation of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 1. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 2. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 1: Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T03:48:56Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 1. Logical design of the molecular computer.]]]]

[[Image:Benenson_fig_2.jpg|thumb|[[Figure 2. Operation of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 1. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 2. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 1: Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T03:48:41Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 1. Logical design of the molecular computer.]]]]

[[Image:Benenson_fig_2_.gif|thumb|[[Figure 2. Operation of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 1. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 2. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 1: Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T03:46:51Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 1. Logical design of the molecular computer.]]]]

[[Image:Benenson_fig_2.gif|thumb|[[Figure 2. Operation of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 1. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 2. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 1: Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T03:46:39Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 1. Logical design of the molecular computer.]]]]

[[Image:Benenson_fig_2.gif|thumb[[Figure 2. Operation of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 1. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 2. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 1: Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T03:44:53Z

Emgarren: /* An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004) */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 1. Logical design of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 1. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 2. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 1: Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

Logic Gates - Emma Garren

2007-12-06T03:42:58Z

Emgarren: /* Logic Gates in Synthetic Biology */

=Background: Logic Gates and Truth Tables=

A [http://en.wikipedia.org/wiki/Logic_gate logic gate], the building block for digital circuits, is a computing unit that performs a logical operation on one or more inputs and produces a single output. Gates are identified by their function, and each type of logic gate can be represented with a distinctive symbol. Inputs to outputs are read from left to right. A small circle on the right indicates the inversion of the output. A double line on the left indicates that the function is "exclusive."

A [http://en.wikipedia.org/wiki/Truth_table truth table] is a useful way to describe the behavior or function of a logic gate. Logic states for both inputs and outputs are designated with 0's and 1's. Click [[Logic Gates: Symbols and Truth Tables|here]] to see some common logic gates and their truth tables.

Logic gates can be placed in parallel or serial combinations in order to perform more complex functions. Here, two parallel outputs, D and E, act as inputs to the function of output Q:

Q = D OR E = (NOT(A OR B)) OR (B AND C)

[[Image:combination_gates.gif]]

([http://www.kpsec.freeuk.com/gates.htm Image] by John Hewes, 2007)

=Logic Gates in Synthetic Biology=
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Adrianantoandro et al., 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are [[Term paper wiki|gene expression noise]], mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Adrianantoandro et al., 2006).

While for digital logic, inputs are either on or off (1 or 0), biological logic is sometimes leads to intermediate induction levels (Voigt, 2006). However, due to their [http://en.wikipedia.org/wiki/Sigmoid_function sigmoid-shaped] dose response curves, gene regulation systems can be considered genetic analog-digital converters. The signal is either ON or OFF for a wide range of input concentrations, with the system changing between the ON and OFF states in a relatively small concentration window (Kramer et al., 2004).

In synthetic biology, logic gates are created by engineering the biochemical reactions that regulate various cellular processes, such as transcription, translation, protein phosphorylation, allosteric regulation, ligand/receptor binding, and enzymatic reactions. Although the diversity of biochemical reactions can make it difficult to combine different devices, these logic gates can be used to build complex systems with functions that have many practical applications. [http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:Mathematical_Models Mathematical modeling] is used to predict the dynamics of the signaling and regulatory networks resulting from the logic gates.

=Biomolecular Logic Gates:'' In Vitro''=

''In vitro'' studies have been used to design combinations of molecules that have emergent properties related to information processing--molecular computing devices. Both the inputs and outputs consist of molecular species, with the output being a biologically active molecule. The extent to which these devices will be used with the cellular context is unclear--however, they are bound to inspire new directions for research in synthetic biology, and have potential applications in biochemical sensing, pathway engineering, and medical diagnosis and treatment.

==Protein-based Logic Gates==

Enzymes:
*Two coupled enzymes perform in parallel the 'AND' and 'InhibAND' logic gate operations. (Baron et al., 2006) Summary:
*Logic Gates and Elementary Computing by Enzymes. (Baron et al., 2006) Summary:

Synthetic Peptide Networks:
*Boolean Logic Functions of a Synthetic Peptide Network. (Ashkenasy and Ghadiri, 2004) Summary:

Signaling Proteins:
*Engineering synthetic signaling proteins with ultrasensitive input/output control. (Dueber et al., 2007) Summary:

==Deoxyribozyme-based Logic Gates==

Deoxyribozyme-based gates are controlled by oligonucleotide inputs, and have been used to engineer logic gates that perform:
*multiple logical operations in parallel
*single-step signaling cascades
*a feedback cycle that acts as an exponential chain reaction

These papers provide examples of deoxyribozyme-based logic gates:
*Deoxyribozyme-Based Ligase Logic Gates and Their Initial Circuits. (Stojanovic et al., 2005) Summary:
*Deoxyribozyme-Based Three-Input Logic Gates and Construction of a Molecular Full Adder. (Lederman et al., 2006) Summary:
*Construction of Molecular Logic Gates with a DNA-Cleaving Deoxyribozyme (Chen et al., 2006) Summary:

==DNA-based Logic Gates==

*Modular Multi-Level Circuits from Immobilized DNA-Based Logic Gates. (Frezza et al., 2007) Summary:
*DNA Logic Gates Based on Structural Polymorphism of Telomere DNA Molecules Responding to Chemical Input Signals. (Miyoshi et al., 2006) Summary:
*Photonic boolean logic gates based on DNA aptamers. (Yoshida and Yokobayashi, 2006) Summary:
*Parallel Molecular Computations of Pairwise Exclusive-OR (XOR) Using DNA "String Tile" Self-Assembly. (Yan et al., 2003) Summary:

==An autonomous molecular computer for logical control of gene expression. (Benenson et al., 2004)==

[[Image:Benenson_fig_1_small.jpg|thumb|[[Figure 1. Logical design of the molecular computer.]]]]

Overview:

This gate uses sequence recognition is used to control enzyme catalysis of covalent bond formation and breakage.
*Input: Specific combination mRNA levels, serving as a simple model of a disease state (cancer)
*Output: “Drug” (or drug repressor) in the form of a single-stranded DNA sequence with known anti-cancer activity

An ''in vitro'' system is designed to recognize a specific combination of mRNA levels as its inputs, and performs a logical operation that results in the production of a molecule that can affect gene expression. The input mRNA levels have been designed to mimic a simplistic version of gene expression modeling cancer, and the output is a drug-like ssDNA with known anticancer activity. Therefore, the molecular computer is analogous to "a computational version of 'diagnosis'" (424).

[[Figure 1. Logical design of the molecular computer.]]

The molecular computers consist of a double-stranded DNA sequence with unique 7-bp sequences that recognize the “input” RNA. Each mRNA indicator is processed one at a time. An “inactivation tag” results in the displacement of the transition molecule, which destroys the computation fragment. An “activation tag” results in activation of the transition molecule. There are two separate molecular computers working simultaneously—one that releases the drug (a specific single-stranded DNA sequence) upon “positive diagnosis,” and another that releases the drug suppressor in response to “negative diagnosis.” The drug (or drug repressor) is incorporated into the DNA fragment as an inactive loop, protected by the double-stranded recognition sequences. If all of the transitions are “positive,” the ultimate output is drug administration (release of ssDNA). If any of the steps results in a “negative” transition, the output is a drug repressor. The design is flexible in that any sufficiently long mRNA molecule can be used as an indicator, and any ssDNA or short RNA molecule (up to at least 21-bp) can be designed as the output. PCR was used to for the experimental demonstration of both diagnosis and drug administration, and it is shown that the amount of active drug increases with the confidence of positive diagnosis. Future work could test the effectiveness of using alternate inputs (such as proteins), alternate outputs (such as RNA interference), as well as testing the function of the molecular computer ''in vivo''.

[[Figure 2. Operation of the molecular computer.]]

==Enzyme-Free Nucleic Acid Logic Circuits. (Seelig et al., 2006)==

Overview:

*Input: 2 DNA strands - F(in) and G(in)
*Gate: 3 DNA strands - E(q), F(f), and G
*Output: Fluorescence - release of F(f)

Nucleic acid devices are simplified by the predicability of base pairing. Although previous research has engineered nucleic acid logic switches based on hybridization and conformational changes ''in vivo'', and this paper designs chemical logic gates that are capable of being combined into large, reliable circuits. These logic gates embody the following digital design principles: logic, cascading, restoration, fan-out, and modularity.

Benefits of this approach: both inputs and outputs are in the same form, which makes cascading possible (the output for one gate serves as the input for the next gate in the circuit).

Gate function is entirely determined by base pairing and breaking. Each gate is composed by one or more gate strands, and one output strand.

[[Figure 1: Two-input AND gate.]]

Restoration:
*When a gate fails to produce enough output when triggered, restoration increases a moderate output amount to the full activation level.
*When a gate "leaks" by spontaneously releasing the output strand, restoration decreases the small output amount to a negligible level.
*Gates for [[Restoration: Amplification Gate|amplification]] and [[Restoration: Thresholding Gate|thresholding]] were used to implement signal restoration.

Modularity and Scalability:
*Eleven gates (AND, OR, sequence translation, input amplification, and signal restoration) were used to compose a large, complex circuit.

=Cellular Logic Gates: ''In Vivo''=

Introductory paragraph here.

==Synthetic Oscillators and Switches==
[[Image:design_fig_1ab.gif|Synthetic oscillators and switches.]]

[[Image:design_fig_1d.gif]]

(Image from Figure 1 of Drubin et al., 2007. Permission pending.)

A. [[Repressilator]] (Elowitz and Leibler, 2000). The "repressilator," or synthetic cellular oscillator, can be built from a string of three repressors, each acting the repress the expression of the next gene in the circuit. Oscillatory output is read by GFP expression regulated by one of the repressors (in this case, tetracycline). The design is analogous to a series of three NOT gates.

B. Toggle switch (Gardner et al., 2000). A toggle switch can be designed from two repressor systems that cross-regulate each other's promoters, and is analogous to the construction of two independent IF gates. It is useful as a pathway module to create more complex programmable cells. Toggle switches can be used to engineer [[CellularMemory:Toggle_Switch|cellular memory]].

D. RNA-based antiswitch. When the engineered RNA is bound to an inducer ligand, the structure changes to either expose or hide a region of RNA that is homologous to a region of the target mRNA (encompassing the translational start site). Thus, when the antisense region is exposed, translation of target mRNA is repressed. Synthetic switches in both eukaryotes ([[Antiswitches|antiswitches]]) and prokaryotes ([[Riboregulators|riboregulators]] and [[Riboswitches|riboswitches]]) can be mediated via RNA devices.

==Environmental signal integration by a modular AND gate. (Anderson et al., 2007)==

AND gates allow cells to integrate multiple signals and can increase their specificity in sensing the environment. This is especially useful for engineering cells to sense environments that are not encountered naturally, or those that are too specific to be identified by a single environmental signal.

A logic gate achieves modularity when it can be used as a self-contained component of more complex systems, and is designed to interface with multiple different inputs and outputs. This paper demonstrates a modular AND gate in ''E. coli'' that uses promoters for both the inputs and the output. This design, unlike that of previously engineered prokaryotic logic gates, makes it relatively easy to use the same gate for different inputs and outputs.

Overview:
*Inputs:
# Promoter that drives the expression of the gene for T7 RNA polymerase (''T7ptag''), containing two [http://en.wikipedia.org/wiki/Stop_codon amber stop codons] that prevent translation under normal circumstances.
#Promoter that drives the expression of the gene for the SupD amber suppressor (''supD''), which decodes the amber stop codons as serine, and allows for translation of T7 RNA polymerase.
*Output: Gene expressed under the T7 promoter, which requires functional translation of both T7 RNA polymerase and the SupD amber suppressor.

The fluorescence data (see [Figure 2. Function of the AND gate. Figure 2]) was used to parameterize a [[transfer function model]] that was derived in order to understand how the range of the input promoters affects the function of the circuit.

[[Figure 1. Schematic representation of AND gate.]]

*Input 1: P(sal) controls the expression supD gene, and is activated by the addition of salicylate (Sal).
*Input 2: P(BAD) controls T7 RNA polymerase gene, and is activated by the addition of arabinose (Ara). Because a strong RBS for T7ptag resulted in high levels of basal expression, the RBS was mutagenized and tuned by screening for the presence of output only when both inducers were present.
*Output: ''GFPmut3_LAA'' (fast-folding GFP with a degradation tag)

[[Figure 2. Function of the AND gate.]]

There is 1000-fold induction of fluorescence in the presence of high concentrations of both inducers, arabinose and salicylate.

[[Figure 6. Modularity of the AND gate.]]

The modularity of the AND gate is demonstrated by reconnecting the gate to new inputs (natural promoters), and a new output (desired phenotype).

A. Exchanging the inputs:
*Input 1: The ''lux'' promoter, which responds to the [[Quorum Sensing|quorum signal]] AI-1.
*Input 2: The ''mgrB'' promoter, which responds to the absence of exogenous magnesium via the PhoPQ two-component system.

B. Exchanging the output:
*Output: The ''inv'' gene, coding for invasin, a protein that allows bacteria to invade mammalian cells.

==BioLogic Gates Enable Logical Transcription Control in Mammalian Cells. (Kramer et al., 2004)==

Transcription control modules, responsive to up to three small molecule inputs, were engineered in mammalian Chinese hamster ovary cells. These "BioLogic gates" provide the tools and building blocks to engineer more complex gene regulatory networks in eukaryotic cells. The gates use butyrolactone-, streptogramin-, tetracycline-, and macrolide-dependent transcription factors, each fused to a KRAB or VP16 repression domain.

#[[NOT IF gate]] - two inputs
#[[NOT IF IF gate]] - three inputs
#[[NAND gate]] - parallel arrangement of two NOT gates
#[[OR gate]] - parallel arrangement of two IF gates
#[[NOR gate]] - constructed from two NOT gates in consecutive order
#[[INVERTER gate]] - combination of two independent IF gates, acts as the inverse of the NOT IF gate

==Cellular Logic with Orthogonal Ribosomes. (Rackham and Chin, 2006)==

Overview:

Two-input logic gates were constructed based on the interactions between synthetic O-rRNA and O-mRNA.
*Input: Orthogonal ribosomes (O-ribosomes), which translate O-mRNA, but do not significantly translate any of the thousands of cellular transcripts bearing cellular RBS sequences.
*Gate: Orthogonal mRNAs (O-mRNAs), which contain ribosome-binding sequences (RBSs) that do not direct the translation of downstream genes by endogenous ribosomes.
*Output: Fluorescence.

Three distinct O-ribosome-O-mRNA pairs were isolated, with molecular specificities for independent function. This was accomplished through [[Hunter Stone - Synthetic Biology Seminar|directed evolution]] in two steps:
#A library of new potential RBSs were placed upstream of a novel fusion of the genes encoding chloramphenicol acetyltransferase and uracil phosphoribosyltransferase, and screened against 5-fluorouracil to select for the O-mRNA sequences that were not translated by endogenous ribosomes.
#The selected O-mRNAs were combined with a library of mutated 16s rRNA sequences, and these cells were grown in the presence of chloramphenicol to screen for those in which the mutant ribosomes translated the O-mRNAs.
The O-ribosome-O-mRNA pairs can be used to control almost any molecular interaction that can be linked to gene expression. In this case they were used to build an AND gate composed of two O-mRNA sequences: O-mRNA-A-omega, encoding the omega fragment of beta-galactosidase, and O-mRNA-C-alpha, encoding the alpha fragment of beta-galactosidase. Synthesis and assembly of a complete beta-galactosidase enzyme (both fragments) results in the cells hydrolyzing FDG into F (fluorescein), which is detected with a fluorometer. Cells programmed with both of the corresponding O-ribosome inputs, O-rRNA-A and O-rRNA-C, exhibited a 20-fold increase in fluorescence when compared with cells containing any other rRNA combinations.

[[Figure 1. O-ribosomes and Boolean logic.]]

=Applications and Future Directions=

Logic gates can be used to design increasingly complex circuits with far-reaching applications in:
*Genetic engineering
*Nanotechnology
*Industrial Fermentation
*Metabolic engineering: increasingly complex synthetic gene circuits might be used to engineer and optimize novel metabolic pathways.
*[[Medical Applications of Synthetic Biology - Samantha Simpson|Medicine]]
**Bacteria to deliver cancer treatment: the integration of multiple inputs can help bacteria sense and respond to increasingly specific environments, such as that of a tumor in the human body.
**Pharmaceuticals: produced through metabolic engineering; "smart" drug delivery.

=References=
*Anderson, J. C., Voigt, C. A., Arkin, A. P. (2007). Environmental signal integration by a modular AND gate. Mol Syst Biol. 3:133. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17700541&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Andrianantoandro, E., Subhayu, B., Karig, D. K., Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Mol Syst. Biol. 2:2006.0028. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16738572&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Ashkenasy, G., Ghadiri, M. R. (2004). Boolean Logic Functions of a Synthetic Peptide Network. J Am Chem Soc. 126(36):11140-1. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15355081&ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Two coupled enzymes perform in parallel the “AND” and “InhibAND” logic gate operations. Org Biomol Chem. 4(6): 989-91. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16525539&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Baron, R., Lioubashevski, O., Katz, E., Niazov, T., Willner, I. (2006). Logic gates and elementary computing by enzymes. J Phys Chem A. 110(27):8548-53. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16821840&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Benenson, Y., Binyamin, G., Ben-Dor, U., Adar, R., Shapiro, E. (2004). An autonomous molecular computer for logical control of gene expression. Nature. 429:423-429. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15116117&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Boczko, E., Gedeon, T., Mischaikow, K. (2007). Dynamics of a simple regulatory switch. J Math Biol. 55(5-6):679-719. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17622532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Chen, X., Wang, Y., Liu, Q., Zhang, Z., Fan, C., He, L. (2006). Construction of molecular logic gates with a DNA-cleaving deoxyribozyme. Angew Chem Int Ed Engl. 45(11):1759-62. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16470893&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Davidson, E.A., Ellington, A.D. (2007). Synthetic RNA circuits. Nat Chem Biol. 3(1):23-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173026&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Dueber, J.E., Mirsky, E.A., Lim, W.A. (2007). Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol. 25(6):660-662. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17515908&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Drubin, D. A., Way, J. C., Silver, P. A. (2007). Designing biological systems. Genes Dev. 21(3):242-54. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17289915&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature. 403(6767):335-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659856&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Farfel, J., Stefanovic, D. (2005). Towards practical biomolecular computers using microfluidic deoxyribozyme logic gate networks. University of New Mexico.
*Frezza, B.M., Cockroft, S. L., Ghadiri, M.R. (2007). Modular Multi-level Circuits from Immobilized DNA-Based Logic Gates. J Am Chem Soc. (Epub ahead of print) [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17994734&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Gardner, T.S., Cantor, C.R., Collins, J.J. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature. 403(6767):338-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10659857&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Heinemann, M., Panke, S. (2006). Synthetic biology—putting engineering into biology. Bioinformatics. 22(22):2790-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16954140&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kaznessis, Y. N. (2007). Models for synthetic biology. BMC Syst Biol. 1(1):47. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17986347&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Kramer, B. P., Fischer, C., Fussenegger, M. (2004). BioLogic Gates Enable Transcription Control in Mammalian Cells. Biotechnol Bioeng. 87(4):478-84. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15286985&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Lederman, H., Macdonald, J., Stefanovic, D., Stojanovic, M. N. (2006). Deoxyribozyme-based three-input logic gates and construction of a molecular full adder. Biochemistry. 45(4):1194-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16430215&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Narayanaswamy, R., Ellington, A.D. (2006). Engineering RNA-based circuits. Handb Exp Pharmacol. (173):423-45. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16594629&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus Abstract]
*Rackham, O., Chin, J. W. (2005). Cellular logic with orthogonal ribosomes. JACS 1227:17584-85. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16351070&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Rackham, O., Chin, J.W. (2006) Synthesizing cellular networks from evolved ribosome-mRNA pairs. Biochem Soc Trans. 34(2):328-9. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16545106&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Sayut, D.J., Kambam, P.K., Sun, L. (2007). Engineering and applications of genetic circuits. Mol Biosyst. 3(12):835-840. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18000560&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Seelig G., Soloveichik, D., Zhang, D. Y., Winfree, E., (2006). Enzyme-free nucleic acid logic circuits. Science. 314(5805): 1585-8. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17158324&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Stojanovic, M. N., Semova, S., Kolpashchikov, D., Macdonald, J., Morgan, C., Stefanovic, D. (2005). Deoxyribozyme-based ligase logic gates and their initial circuits. J Am Chem Soc. 127(19):6914-5. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15884910&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Voigt, C. A. (2006). Genetic parts to program bacteria. Curr Opin Biotechnol. 17:548-557. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16978856&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Wall, M. E., Hlavacek, W. S., Savageau, M. A. (2004). Design of gene circuits: lessons from bacteria. Nat Rev Genet. 5(1):34-42. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14708014&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yan, H., Feng, L., LaBean, T.H., Reif, J.H. (2003). Parallel Molecular Computations of Pairwise Exclusive-Or (XOR) Using DNA "String Tile" Self-Assembly. J Am Chem Soc. 125:14246-14247. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14624551&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]
*Yoshida, W., Yokobayashi, Y. (2007). Photon Boolean logic gates based on DNA aptamers. Chem Commun (Camb). (2):195-7. [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17180244&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Abstract]

File:Logic gates table.gif

2007-12-06T03:40:12Z

Emgarren:

Davidson College Synthetic Biology Seminar (Fall 2007)

2007-11-27T21:24:17Z

Emgarren: /* Our Papers */

== Davidson College Synthetic Biology Seminar, Fall 2007 ==
Click [http://www.bio.davidson.edu/Courses/Synthetic/synthetic_Seminar.html here] to access the class webpage

===Synthetic Biology Defined===

===Synthetic Biology: A Brief Introduction===

In 1978, the Nobel Prize in Medicine went to Werner Arber, Daniel Nathans, and Hamilton O. Smith for discovering restriction enzymes (www.nobelprize.org), which launched recombinant DNA technology. Scientists were able to splice open segments of DNA at specific restriction sites and add or take away different DNA segments, seeing how these DNA changes affected cell function. Recombinant DNA technology marks the beginning of the field of synthetic biology.

Using restriction enzymes and recombinant DNA, scientists focused on areas as diverse as biofuels, cellular memory, and directed evolution. Synthetic biology now captures a wide range of disciplines that all hold one thing in common: engineering genomes using preexisting and new biological systems and components. Mathematical modeling informs the design of the synthetic system, device, or circuit, which is implemented in wet lab experiments. Results from experimentation enhance the model. The ultimate goal of synthetic biology, therefore, is not only building novel biological systems, but creating a better understanding of existing ones.

===Synthetic Biology in the Media===

Here we could link to some articles accessible to the general public...

===Our Papers===

Students in Dr. Campbell's Fall 2007 Synthetic Biology Seminar each wrote a paper on a specific topic within the field of synthetic biology. With our selections, we do not claim to cover all aspects of synthetic biology, but instead hope to provide an overview on subjects we found interesting. The topics we chose can be classified under three broader areas of synthetic biology research:

#Designng and Optimizing Tools. Mike, Laura, Hunter
#Small Parts and Devices. Erin, Emma, Danielle
#Broader Applications. Samantha, Will

[[Stochasticity in Gene Expression- Mike Waters]] 
My paper will cover a characterization, implications, and ways to manipulate stochastic processes during gene expression.

[[Post-transcriptional Regulation Technologies - Erin Zwack]]

Using regulatory RNA, gene expression can now be controlled at the stage after transcription but before translation.

[[CellularMemory:Main Page | Synthetic Cellular Memory - Will DeLoache]]

Synthetic cellular memory refers to the engineering of living organisms to produce "a protracted response to a transient stimulus" ([http://gcat.davidson.edu/GcatWiki/index.php/CellularMemory:References Ajo-Franklin, 2007]). The construction of such rationally designed memory mechanisms in living organisms provides a more thorough understanding of naturally occurring gene networks. In the future, modular cellular memory networks will likely be a key component of many synthetic biological designs, ranging from biocomputing to engineered cell differentiation.

[[Medical Applications of Synthetic Biology - Samantha Simpson]]

Medical applications of synthetic biology range from treating cancer, creating low-cost medication, protecting from DNA damage, and using biological vectors as vaccines. My paper explores these current collaborations between medicine and synthetic biology, and the challenges and benefits to come in the future.

[[Logic Gates - Emma Garren]]

Logic gates are computing units that perform a logical function on one or more inputs to produce a single output. Synthetic biologists use various cellular regulation mechanisms (transcription, translation, etc.) to create modular gene expression devices that can be combined in order to engineer cells that perform increasingly complex tasks.

[[Laura Voss - Synthetic Biology Seminar | Promoters and Reporters in Synthetic Biology - Laura Voss]]

Key to the construction of gene circuits and biosensors are promoter and reporter genes, which control how a cell's genes are transcribed when the cell's environment changes. In addition to using promoters and reporters as available to build cellular machines, synthetic biologists can also alter, redesign, or engineer these genetic components in order to refine biological design.

[[Applications of Ribozymes in Synthetic Systems - Danielle Jordan]]

Ribozymes, or RNA enzymes, serve an important role in cellular function both by acting as carriers of genetic infomation and as catalysts for chemical reactions. These enzymes, which represent important ways of regulating genes, that have yet to be fully discovered. Synthetic biology attempts to understand these complex interactions by creating artificial ribozymes and placing them into existing systems. This modular method of gene regulation could open new ways of solving existing promoter and reporter interactions.

[[Directed Evolution and Synthetic Biology - Hunter Stone]]

Directed evolution is a method of cellular engineering that uses Darwinian selection to evolve proteins and RNA with desirable properties not found in nature. The reliance of this method on the randomness of mutation and nature's selective properties sharply contrasts to the logical modeling and reasoning associated with traditional synthetic methods. Some might say that the lack of planning involved with directed evolution means it is constitutionally different than synthetic biology. Regardless, the method has been shown to be effective in achieving desired results in a number of cases, and could prove instrumental in the optimization of synthetically-designed constructs.

===Other Interesting Areas of Research===

Here we could put a brief summary of some other areas of synthetic biology research that are not covered in by any of our papers. These might include:

*Nanotechnology.
*Engineering the minimal cell. Scientists are trying to identify or build a cell that contains only those elements necessary to be able to classify it as "living." Bot top-down (Venter) and bottom-up (Luisi) approaches are being used. This research could shed light on the earliest origins of life, as well as provide a vessel (chassis? I don't know correct use of this word...) for engineering synthetic biological functions that avoids the variables typically encountered when introducing a synthetic pathway into a cell.
*Other ideas?