GcatWiki - User contributions [en]

IGEM 2009 Project

2009-06-04T16:37:23Z

Brs3298:

Biology Based:
#[[Which reporters are we going to use?]]
#[[What naming system are we going to use for the suppressor tRNAs?]]
#[[How do you build the tRNA construct?]]
#[[How are we going to build the 5-mer BioBricks?]]

[http://gcat.davidson.edu/GcatWiki/images/2/2c/Stop_Codons_in_LCs.doc Using Stop Codons to Truncate Translation] 
[http://gcat.davidson.edu/GcatWiki/images/8/81/D5merSequences.doc Davidson ATG+5mer BioBrick Sequences]

[http://gcat.davidson.edu/GcatWiki/images/3/3d/FML_CODING_SEQUENCE.doc Frameshift Mutation Leader PCR primers]

Math Based:

IGEM 2009 Project

2009-06-04T16:37:10Z

Brs3298:

Biology Based:
#[[Which reporters are we going to use?]]
#[[What naming system are we going to use for the suppressor tRNAs?]]
#[[How do you build the tRNA construct?]]
#[[How are we going to build the 5-mer BioBricks?]]

[http://gcat.davidson.edu/GcatWiki/images/2/2c/Stop_Codons_in_LCs.doc Using Stop Codons to Truncate Translation] 
[http://gcat.davidson.edu/GcatWiki/images/8/81/D5merSequences.doc Davidson ATG+5mer BioBrick Sequences]
[http://gcat.davidson.edu/GcatWiki/images/3/3d/FML_CODING_SEQUENCE.doc Frameshift Mutation Leader PCR primers]

Math Based:

File:FML CODING SEQUENCE.doc

2009-06-04T16:35:42Z

Brs3298:

IGEM 2009 Project

2009-06-04T16:34:51Z

Brs3298:

Biology Based:
#[[Which reporters are we going to use?]]
#[[What naming system are we going to use for the suppressor tRNAs?]]
#[[How do you build the tRNA construct?]]
#[[How are we going to build the 5-mer BioBricks?]]

[http://gcat.davidson.edu/GcatWiki/images/2/2c/Stop_Codons_in_LCs.doc Using Stop Codons to Truncate Translation] 
[http://gcat.davidson.edu/GcatWiki/images/8/81/D5merSequences.doc Davidson ATG+5mer BioBrick Sequences]
[Frameshift Mutation Leader PCR Primers]

Math Based:

What naming system are we going to use for the suppressor tRNAs?

2009-06-03T20:38:37Z

Brs3298:

<center> [[Image:naming suppressors.GIF]] </center>

In order to make sure that we are able to talk about any given suppressor tRNA, we are going to need a naming system that everyone can use.

File:Naming suppressors.GIF

2009-06-03T20:35:23Z

Brs3298:

What naming system are we going to use for the suppressor tRNAs?

2009-06-03T20:35:03Z

Brs3298:

<center> [[Image:naming suppressors.GIF]] </center>

In order to make sure that we are able to talk about any given suppressor tRAN, we are going to need a naming system that everyone can use.

What naming system are we going to use for the suppressor tRNAs?

2009-06-03T20:21:10Z

Brs3298:

<center> [[Image:Table2_Anderson2002.GIF]] </center>

IGEM 2009 Project

2009-06-03T20:16:03Z

Brs3298:

IGEM 2009 Project

2009-06-03T20:14:55Z

Brs3298:

Missouri Western/Davidson iGEM2009

2009-06-03T20:14:14Z

Brs3298: /* iGEM 2009 Project */

This space will be used starting April, 2009 for brainstorming and a shared whiteboard space.

[http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Davidson_Protocols Davidson Lab Protocols] 
[http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/MWSU_protocols MWSU Lab Protocols] 
[http://gcat.davidson.edu/sybr-u/bmc.html BioMath Connections Page] 
[http://gcat.davidson.edu/GCATalog-r2.2/GCATalog.htm GCAT-alog Freezer Stocks] 
<center>
== MWSU-Davidson 2009 iGEM team photo ==
[[Image:iGEMTeam2009.png|250px|]] 
== The Davidson College part of the 2009 iGEM Team ==
[[Image:DavidsonTeam2009.png|429px|]]
</center>

= [[iGEM 2009 Project]] =

We need to learn more about these topics:
<center>'''Biology-based'''</center>

#[[Plasmid Creation]]
#[[What is msDNA?]]
#[[How is msDNA normally produced?]] Olivia/Alyndria
#[[How many copies are carried per cell?]] Alyndria
#[[What would we need to do to turn this into a BioBrick device?]] Romina
#[[ How could we swap out msDNA sequences?]] Shamita
#[[What role can physical modeling of protein structure play in our project?]]
#[[What role can physical modeling of proteins play in our project? Eric Sawyer]]
#[[What other cool reporters are there? (Discrete On/Off or Continuous) Bryce Szczepanik]]
#[[Can we use promoter strength/opposite directions to subtract? Clif Davis]]
#[[Can we use suppressor tRNAs to encode logical operators (suppressor suppressor logic, SSL)?]]
#[[How is msDNA stored in E. coli?]] Olivia
#[[What is the sequence of bacterial reverse transcriptase and can we clone that gene?]] Shamita
#[[Can we redesign the normal msDNA pathway to produce new segments of DNA of our choosing?]] All
#[[What are other available reverse transcriptases?]] Leland
#[[What other math problems (e.g. NP- complete) are accessible to us? Annie Siya Sun]]
#[[What is the relationship between 3-SAT and map coloring? Ashley Schnoor]]
#[[What activators are there that turn on a promoter without any help?]]
#[[Can we use protein interactions to compute? (Post-translation, proteases, quaternary structure) Will Vernon]]
#[[Could we do something with clocks/counting?]]
#[[Could we have/use multiple synthetic organelles in a cell?]]
#[[What ideas from previous iGEM teams are useful to us?]]
#[[Examples of Metabolic Pathways in E.coli]]

<center>'''Math-based'''</center>
#[[Can we get bacteria to solve a problem large enough to challenge a person?]]
#[[What interesting challenges or problems does origami offer?]]
#[[Can we produce a series of increasingly difficult goals that might be possible to produce in the lab?]]
#[[What has been done before and how can we improve upon that?]]
#[[We can perform some pilot experiments using synthesized DNA and later switch to msDNA (maybe).]]
#[[Can we address the Boolean Satisfiability (SAT) problem with a bacterial computer?]]
#[[How has 3SAT been addressed with a DNA computer? Can we use those methods?]]

#Can we get bacteria to solve a problem large enough to challenge a computer (probably not, but it is fun to think about)?
#[[What are some linear algebra applications for DNA origami?]]
#[[How can we use origami to solve 3-SAT problems?]]

<center>'''Behavior-based'''</center>
#[[What constructs are we testing?]]
#[[What school districts do we have access to?]]
#[[Where is the Synthetic Biology page we want high school teachers to use after the survey?]]
#[[Do you need any more input from the veterans before the survey is ready?]]

<center>'''Solving The 3SAT Problem Using Suppressor Logic'''</center>
#[[Can we solve a 3-SAT problem with supressor logic?]]

<center>'''Hin/Hix System'''</center>
Here is a link to the animation of the Hin/Hix system created by the "E. HOP living computer project folks.
[http://www.bio.davidson.edu/people/kahaynes/FAMU_talk/Living_computer.swf Hin/Hix Animation]

IGEM 2009 Project

2009-06-03T20:13:29Z

Brs3298:

#[[Which reporters are we going to use?]]
#[[What naming system are we going to use for the suppressor tRNAs?]]
#[[How do you build the tRNA construct?]]
#[[How are we going to build the 5-mer BioBricks?]]

[http://gcat.davidson.edu/GcatWiki/images/2/2c/Stop_Codons_in_LCs.doc Using Stop Codons to Truncate Translation]

Can we solve a 3-SAT problem with supressor logic?

2009-06-03T20:12:56Z

Brs3298: /* How could suppressor logic be used to solve the Sakamoto 3-SAT problem? */

== What is the 3-SAT problem? ==

''Advantages/Disadvantages to using the 3 SAT problem''

Advantages:

1) Seems to have an equal balance of mathematics and biology.

2) Eventually, there is promise to make the problem difficult enough whereas a computer would have a hard time solving it.

3) Introducing a new concept (supressor supressor logic)

Disadvantages:

1) Using the same concept/idea that has been used by previous Missouri Western/Davidson iGEM teams. (XOR gate)

2) Finding enough for everyone to work on for the entire summer.

3) Finding a promoter that is turned on without the use of a small particle.

== How did Sakamoto et al. use a DNA computer to solve a 3-SAT problem? ==

[http://gcat.davidson.edu/GcatWiki/images/3/39/Sakamoto_DNA_computer_3SAT.pdf Sakamoto et al.]

<center> [[Image:hairpin2.GIF]] </center>
Sakamoto used hairpin formations in single stranded DNA (ssDNA) as a molecular computer. Hairpin structures are formed when complimentary bases on the same strand attach to each other forming a loop. As shown in Picture B above, C and -C are compliments of each other, and bind together. Picture A is what a normal ssDNA should look like. Sakamoto used this self assembly of secondary structures on a satisfiability (SAT) problem. The problem had six inputs and ten clauses. An example of a clause would be (a or b or -c), and could include any combinations of inputs from a to f including -a to -f. If the problem is satisfied, the ssDNA stays in it's normal form. If the problem is not satisfied, the ssDNA forms a hairpin. Later in the paper it explains that the hairpin forming molecules can be removed from the others with certain techniques. 

The 10 clause 3-SAT problem solved in the paper is:

(a or b or –c) 
(a or c or d) 
(a or –c or –d) 
(-a or –c or d) 
(a or –c or e) 
(a or d or –f) 
(-a or c or d) 
(a or c or –d) 
(-a or –c or –d) 
(-a or c or –d) 

note: this problem uses inputs of a, b, c, d, e, -a, -c, -d, -f (f, -b, -e are not used)

== What is suppressor logic? ==

Will's Version of the PPT for SSL 3SAT
[http://gcat.davidson.edu/GcatWiki/images/6/6f/Will%27s_SSL_3SAT.ppt Will's Version of the PPT for SSL 3SAT]

Powerpoint on 3SAT SSL (Bryce and Clif) I don't know how to post the powerpoint on here.
[http://gcat.davidson.edu/GcatWiki/images/d/d1/3SAT_ppt-1-.ppt Bryce and Clif's Version]

'''Suppressor Logic uses suppressor tRNAs as inputs to avoid frameshift mutations in the production of an output amino acid sequence'''

A frameshift is a genetic mutation caused by the addition or deletion of nucelotides to a given sequence which codes for a protein. Since codons are read in a series of three, the addition or deletion of nucleotides will disrupt the reading frame. This disruption will most likely cause the production of a nonfunctional protein.

<center> [[Image:suppressor4.GIF]] </center>

A frameshift occurs and, in this case, a guanine is added to the sequence. If nothing is done, enzyme A will not be made, meaning the clause will not be satisfied.

The suppressor tRNA allows the 4 letter sequence to be read as a single codon, therefore, keeping the protein on track. 

If more than one frameshift mutation is introduced into a coding sequence, then logical operators can be encoded. Suppressor a binds to CCCG, supressor b binds to CUGC, and suppressor c binds to ACCG below: 

# AUG CCCG CUG ... rest of Gene 
# AUG CCCG CUGC CUG ... rest of Gene 
# AUG CCCG gg CUGC AGG ... rest of Gene 
# AUG CCCG gg CUGC gg ACCG AGG ... rest of Gene 
 
{| align="center" border="1" cellpadding="8" cellspacing="0" style="background:lightcyan; font-weight:bold; text-align:center; width:45%"
|+
|- style="background:paleturquoise"
! style="width:15%" | Construct
! style="width:15%" | Gene
! style="width:15%" | Logical operation
|-
| 1 || Phenotype || a
|-
| 2 || Phenotype || a AND b
|-
| 3 || Phenotype || a OR b
|-
| 4 || Phenotype || a OR b OR c
|-
| 1 || Repressor|| NOT a
|-
| 2 || Repressor|| a NAND b
|-
| 3 || Repressor|| a NOR b
|-
| 4 || Repressor|| NOT (a OR b OR c)
|}

'''Logical clauses can be connected by AND operators if the proteins produced are part of a biochemical pathway.''' In this case, a AND b AND c: 

<center> [[Image:suppressor2.GIF]] </center>

== How could suppressor logic be used to solve the Sakamoto 3-SAT problem? ==

'''Definitions'''

Inputs = framshift suppressor tRNAs

Input value = supp a is 1, supp g is 0; supp b is 1, supp h is 0, etc. up to tth 6th pair of f and l

Logical clause (LC) = three inputs connected by OR, eg. (a OR b OR e)

LC using 4-base frameshift mutations: cag CCCG aa GGGC tt GTTG cag (may also have any multiple of 3 bases between the mutations) 

LC using 5-base frameshift mutations: cag CCCGC a GGGCG t GTTGC cag (may also have any multiple of 3 bases between the mutations) 

This makes an XOR logic gate as only one suppressor can be used at one time to maintain the reading frame.

Logical expression (LE) = string of LCs connected by AND 
The design below encodes LC1 AND LC2 AND LC3 

<center> [[Image:logical expression2.GIF]] </center>

'''Subroutine'''

1. Individual bacteral cells use Hin/hix system to randomly choose of of the 64 possible combinations of 6 inputs. Suppressors a and g represent 1 and 0 for the first input; suppressors b and h are 1 and 0 for the second input; etc. up to the sixth input with suppressors f and l (lower case L). The triangles are hix sites for Hin recombination. Whichever of the two suppressor tRNAs in an input pair is facing forward determines whether the value of that input is 1 or 0.

<center> [[Image:suppressor inputs.GIF]] </center>

2. Each bacterial cell carries out the following subroutine on each LC: IF LC=TRUE THEN "check the next LC" ELSEIF LC=FALSE "go get a new set of inputs with step 1" In order to do this, each of the activators below must turn on a repressor that turns off Hin production. Then if one of the activators is not made, Hin will be made, and new inputs will be established.

<center> [[Image:LE circuit.GIF]] </center>

3. If/when a bacterial cell finds a set of inputs that satisfies the entire LE (ie. a solution to the 3-SAT problem), it will glow green.

== Things we need to learn about ==

'''Note: We would need 12 different frameshift suppressor tRNAs to encode the Sakamoto 3-SAT problem'''

'''Discovery of frameshift tRNAs. How many are known?''' 

[http://gcat.davidson.edu/GcatWiki/images/5/59/MaglierySchultz2001JMB.pdf Magliery, Anderson, Schultz]

Library approach used to discover efficient suppressors of four-base codons AGGA, UAGA, CCCU, and CUAG using mutated versions of serine tRNA.

[http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf Anderson, Maglieri, Schultz]

Signals for translational bypassing (slipping and hopping): mRNA secondary structure, "hungry" (underused) codons, upstream Shine-Dalgarno-like (RBS) sequences 
Library approach extended in order to discover frameshift suppressor tRNAs with anticodons of size two to six bases. Two-base and six-base suppressors were not found, but the following five-base suppressors were found:

[http://gcat.davidson.edu/GcatWiki/images/e/e0/PNAS-2006-Rodriguez-8650-5.pdf Rodriguez, Lester, Dougherty]

[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T36-4HM7N2R-G&_user=2665120&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000058476&_version=1&_urlVersion=0&_userid=2665120&md5=d2dda7b99095c256fd459f2f6c82b493 3 Suppressor tRNAs in a single protein, in vitro]

[http://jb.asm.org/cgi/content/abstract/158/3/849 Three suppressor tRNAs in vivo (E. coli)]

Incorporating unnatural amino acids using frameshift suppression. They used three in one eukaryotic cell.
<center> [[Image:Table2_Anderson2002.GIF]] </center>

[http://gcat.davidson.edu/GcatWiki/images/a/ac/Dunham_2009_tRNA_structure.pdf Dunham et al.]

[http://gcat.davidson.edu/GcatWiki/images/c/c3/Hohsaka_2009_in_vitro_tln.pdf Hohsaka et al.]

'''Processing of tRNA precursors in E. coli''' 

[http://www.nature.com/embor/journal/v2/n1/full/embor501.html Mörl and Marchfelder] describe processing 

<center> [[Image:the_final_cut.GIF]] </center>

'''How many different frameshift suppressor tRNAs can be in one cell without causing toxicity?'''

Can we have twelve of them? If not, what is the maximum? Is there an impact of codon usage on this?

Can we have both 4- and 5-base suppressor tRNAs in one cell?

Are there particular combinations of suppressors that are more tolerated than others?

 
'''How many frameshift mutations can be suppressed in a single gene?'''

How close together can the frameshift mutations of a given LC be?

Can we mix 4- and 5-base frameshift mutations in a given LC?

IGEM 2009 Project

2009-06-03T20:05:29Z

Brs3298:

Missouri Western/Davidson iGEM2009

2009-06-03T20:00:25Z

Brs3298: /* iGEM 2009 Project */

This space will be used starting April, 2009 for brainstorming and a shared whiteboard space.

[http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Davidson_Protocols Davidson Lab Protocols] 
[http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/MWSU_protocols MWSU Lab Protocols] 
[http://gcat.davidson.edu/sybr-u/bmc.html BioMath Connections Page] 
[http://gcat.davidson.edu/GCATalog-r2.2/GCATalog.htm GCAT-alog Freezer Stocks] 
<center>
== MWSU-Davidson 2009 iGEM team photo ==
[[Image:iGEMTeam2009.png|250px|]] 
== The Davidson College part of the 2009 iGEM Team ==
[[Image:DavidsonTeam2009.png|429px|]]
</center>

== [[iGEM 2009 Project]] ==

We need to learn more about these topics:
<center>'''Biology-based'''</center>

#[[Plasmid Creation]]
#[[What is msDNA?]]
#[[How is msDNA normally produced?]] Olivia/Alyndria
#[[How many copies are carried per cell?]] Alyndria
#[[What would we need to do to turn this into a BioBrick device?]] Romina
#[[ How could we swap out msDNA sequences?]] Shamita
#[[What role can physical modeling of protein structure play in our project?]]
#[[What role can physical modeling of proteins play in our project? Eric Sawyer]]
#[[What other cool reporters are there? (Discrete On/Off or Continuous) Bryce Szczepanik]]
#[[Can we use promoter strength/opposite directions to subtract? Clif Davis]]
#[[Can we use suppressor tRNAs to encode logical operators (suppressor suppressor logic, SSL)?]]
#[[How is msDNA stored in E. coli?]] Olivia
#[[What is the sequence of bacterial reverse transcriptase and can we clone that gene?]] Shamita
#[[Can we redesign the normal msDNA pathway to produce new segments of DNA of our choosing?]] All
#[[What are other available reverse transcriptases?]] Leland
#[[What other math problems (e.g. NP- complete) are accessible to us? Annie Siya Sun]]
#[[What is the relationship between 3-SAT and map coloring? Ashley Schnoor]]
#[[What activators are there that turn on a promoter without any help?]]
#[[Can we use protein interactions to compute? (Post-translation, proteases, quaternary structure) Will Vernon]]
#[[Could we do something with clocks/counting?]]
#[[Could we have/use multiple synthetic organelles in a cell?]]
#[[What ideas from previous iGEM teams are useful to us?]]
#[[Examples of Metabolic Pathways in E.coli]]

<center>'''Math-based'''</center>
#[[Can we get bacteria to solve a problem large enough to challenge a person?]]
#[[What interesting challenges or problems does origami offer?]]
#[[Can we produce a series of increasingly difficult goals that might be possible to produce in the lab?]]
#[[What has been done before and how can we improve upon that?]]
#[[We can perform some pilot experiments using synthesized DNA and later switch to msDNA (maybe).]]
#[[Can we address the Boolean Satisfiability (SAT) problem with a bacterial computer?]]
#[[How has 3SAT been addressed with a DNA computer? Can we use those methods?]]

#Can we get bacteria to solve a problem large enough to challenge a computer (probably not, but it is fun to think about)?
#[[What are some linear algebra applications for DNA origami?]]
#[[How can we use origami to solve 3-SAT problems?]]

<center>'''Behavior-based'''</center>
#[[What constructs are we testing?]]
#[[What school districts do we have access to?]]
#[[Where is the Synthetic Biology page we want high school teachers to use after the survey?]]
#[[Do you need any more input from the veterans before the survey is ready?]]

<center>'''Solving The 3SAT Problem Using Suppressor Logic'''</center>
#[[Can we solve a 3-SAT problem with supressor logic?]]

<center>'''Hin/Hix System'''</center>
Here is a link to the animation of the Hin/Hix system created by the "E. HOP living computer project folks.
[http://www.bio.davidson.edu/people/kahaynes/FAMU_talk/Living_computer.swf Hin/Hix Animation]

Missouri Western/Davidson iGEM2009

2009-06-03T19:59:41Z

Brs3298: /* iGEM 2009 Project */

This space will be used starting April, 2009 for brainstorming and a shared whiteboard space.

[http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Davidson_Protocols Davidson Lab Protocols] 
[http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/MWSU_protocols MWSU Lab Protocols] 
[http://gcat.davidson.edu/sybr-u/bmc.html BioMath Connections Page] 
[http://gcat.davidson.edu/GCATalog-r2.2/GCATalog.htm GCAT-alog Freezer Stocks] 
<center>
== MWSU-Davidson 2009 iGEM team photo ==
[[Image:iGEMTeam2009.png|250px|]] 
== The Davidson College part of the 2009 iGEM Team ==
[[Image:DavidsonTeam2009.png|429px|]]
</center>

== iGEM 2009 Project ==

We need to learn more about these topics:
<center>'''Biology-based'''</center>

#[[Plasmid Creation]]
#[[What is msDNA?]]
#[[How is msDNA normally produced?]] Olivia/Alyndria
#[[How many copies are carried per cell?]] Alyndria
#[[What would we need to do to turn this into a BioBrick device?]] Romina
#[[ How could we swap out msDNA sequences?]] Shamita
#[[What role can physical modeling of protein structure play in our project?]]
#[[What role can physical modeling of proteins play in our project? Eric Sawyer]]
#[[What other cool reporters are there? (Discrete On/Off or Continuous) Bryce Szczepanik]]
#[[Can we use promoter strength/opposite directions to subtract? Clif Davis]]
#[[Can we use suppressor tRNAs to encode logical operators (suppressor suppressor logic, SSL)?]]
#[[How is msDNA stored in E. coli?]] Olivia
#[[What is the sequence of bacterial reverse transcriptase and can we clone that gene?]] Shamita
#[[Can we redesign the normal msDNA pathway to produce new segments of DNA of our choosing?]] All
#[[What are other available reverse transcriptases?]] Leland
#[[What other math problems (e.g. NP- complete) are accessible to us? Annie Siya Sun]]
#[[What is the relationship between 3-SAT and map coloring? Ashley Schnoor]]
#[[What activators are there that turn on a promoter without any help?]]
#[[Can we use protein interactions to compute? (Post-translation, proteases, quaternary structure) Will Vernon]]
#[[Could we do something with clocks/counting?]]
#[[Could we have/use multiple synthetic organelles in a cell?]]
#[[What ideas from previous iGEM teams are useful to us?]]
#[[Examples of Metabolic Pathways in E.coli]]

<center>'''Math-based'''</center>
#[[Can we get bacteria to solve a problem large enough to challenge a person?]]
#[[What interesting challenges or problems does origami offer?]]
#[[Can we produce a series of increasingly difficult goals that might be possible to produce in the lab?]]
#[[What has been done before and how can we improve upon that?]]
#[[We can perform some pilot experiments using synthesized DNA and later switch to msDNA (maybe).]]
#[[Can we address the Boolean Satisfiability (SAT) problem with a bacterial computer?]]
#[[How has 3SAT been addressed with a DNA computer? Can we use those methods?]]

#Can we get bacteria to solve a problem large enough to challenge a computer (probably not, but it is fun to think about)?
#[[What are some linear algebra applications for DNA origami?]]
#[[How can we use origami to solve 3-SAT problems?]]

<center>'''Behavior-based'''</center>
#[[What constructs are we testing?]]
#[[What school districts do we have access to?]]
#[[Where is the Synthetic Biology page we want high school teachers to use after the survey?]]
#[[Do you need any more input from the veterans before the survey is ready?]]

<center>'''Solving The 3SAT Problem Using Suppressor Logic'''</center>
#[[Can we solve a 3-SAT problem with supressor logic?]]

<center>'''Hin/Hix System'''</center>
Here is a link to the animation of the Hin/Hix system created by the "E. HOP living computer project folks.
[http://www.bio.davidson.edu/people/kahaynes/FAMU_talk/Living_computer.swf Hin/Hix Animation]

Missouri Western/Davidson iGEM2009

2009-06-03T19:59:21Z

Brs3298: /* The Davidson College part of the 2009 iGEM Team */

This space will be used starting April, 2009 for brainstorming and a shared whiteboard space.

[http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Davidson_Protocols Davidson Lab Protocols] 
[http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/MWSU_protocols MWSU Lab Protocols] 
[http://gcat.davidson.edu/sybr-u/bmc.html BioMath Connections Page] 
[http://gcat.davidson.edu/GCATalog-r2.2/GCATalog.htm GCAT-alog Freezer Stocks] 
<center>
== MWSU-Davidson 2009 iGEM team photo ==
[[Image:iGEMTeam2009.png|250px|]] 
== The Davidson College part of the 2009 iGEM Team ==
[[Image:DavidsonTeam2009.png|429px|]]
</center>

=== iGEM 2009 Project ===

We need to learn more about these topics:
<center>'''Biology-based'''</center>

#[[Plasmid Creation]]
#[[What is msDNA?]]
#[[How is msDNA normally produced?]] Olivia/Alyndria
#[[How many copies are carried per cell?]] Alyndria
#[[What would we need to do to turn this into a BioBrick device?]] Romina
#[[ How could we swap out msDNA sequences?]] Shamita
#[[What role can physical modeling of protein structure play in our project?]]
#[[What role can physical modeling of proteins play in our project? Eric Sawyer]]
#[[What other cool reporters are there? (Discrete On/Off or Continuous) Bryce Szczepanik]]
#[[Can we use promoter strength/opposite directions to subtract? Clif Davis]]
#[[Can we use suppressor tRNAs to encode logical operators (suppressor suppressor logic, SSL)?]]
#[[How is msDNA stored in E. coli?]] Olivia
#[[What is the sequence of bacterial reverse transcriptase and can we clone that gene?]] Shamita
#[[Can we redesign the normal msDNA pathway to produce new segments of DNA of our choosing?]] All
#[[What are other available reverse transcriptases?]] Leland
#[[What other math problems (e.g. NP- complete) are accessible to us? Annie Siya Sun]]
#[[What is the relationship between 3-SAT and map coloring? Ashley Schnoor]]
#[[What activators are there that turn on a promoter without any help?]]
#[[Can we use protein interactions to compute? (Post-translation, proteases, quaternary structure) Will Vernon]]
#[[Could we do something with clocks/counting?]]
#[[Could we have/use multiple synthetic organelles in a cell?]]
#[[What ideas from previous iGEM teams are useful to us?]]
#[[Examples of Metabolic Pathways in E.coli]]

<center>'''Math-based'''</center>
#[[Can we get bacteria to solve a problem large enough to challenge a person?]]
#[[What interesting challenges or problems does origami offer?]]
#[[Can we produce a series of increasingly difficult goals that might be possible to produce in the lab?]]
#[[What has been done before and how can we improve upon that?]]
#[[We can perform some pilot experiments using synthesized DNA and later switch to msDNA (maybe).]]
#[[Can we address the Boolean Satisfiability (SAT) problem with a bacterial computer?]]
#[[How has 3SAT been addressed with a DNA computer? Can we use those methods?]]

#Can we get bacteria to solve a problem large enough to challenge a computer (probably not, but it is fun to think about)?
#[[What are some linear algebra applications for DNA origami?]]
#[[How can we use origami to solve 3-SAT problems?]]

<center>'''Behavior-based'''</center>
#[[What constructs are we testing?]]
#[[What school districts do we have access to?]]
#[[Where is the Synthetic Biology page we want high school teachers to use after the survey?]]
#[[Do you need any more input from the veterans before the survey is ready?]]

<center>'''Solving The 3SAT Problem Using Suppressor Logic'''</center>
#[[Can we solve a 3-SAT problem with supressor logic?]]

<center>'''Hin/Hix System'''</center>
Here is a link to the animation of the Hin/Hix system created by the "E. HOP living computer project folks.
[http://www.bio.davidson.edu/people/kahaynes/FAMU_talk/Living_computer.swf Hin/Hix Animation]

Missouri Western/Davidson iGEM2009

2009-06-01T16:40:18Z

Brs3298: /* Davidson College 2009 iGEM Team */

This space will be used starting April, 2009 for brainstorming and a shared whiteboard space.

[http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Davidson_Protocols Davidson Lab Protocols] 
[http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/MWSU_protocols MWSU Lab Protocols] 
[http://gcat.davidson.edu/sybr-u/bmc.html BioMath Connections Page] 
[http://gcat.davidson.edu/GCATalog-r2.1/GCATalog.htm GCAT-alog Freezer Stocks] 
<center>
== MWSU-Davidson 2009 iGEM team photo ==
[[Image:iGEMTeam2009.png|250px|]] 
== The Davidson College part of the 2009 iGEM Team ==
[[Image:DavidsonTeam2009.png|429px|]]
</center>

We need to learn more about these topics:
<center>'''Biology-based'''</center>

#[[Plasmid Creation]]
#[[What is msDNA?]]
#[[How is msDNA normally produced?]] Olivia/Alyndria
#[[How many copies are carried per cell?]] Alyndria
#[[What would we need to do to turn this into a BioBrick device?]] Romina
#[[ How could we swap out msDNA sequences?]] Shamita
#[[What role can physical modeling of protein structure play in our project?]]
#[[What role can physical modeling of proteins play in our project? Eric Sawyer]]
#[[What other cool reporters are there? (Discrete On/Off or Continuous) Bryce Szczepanik]]
#[[Can we use promoter strength/opposite directions to subtract? Clif Davis]]
#[[Can we use suppressor tRNAs to encode logical operators (suppressor suppressor logic, SSL)?]]
#[[How is msDNA stored in E. coli?]] Olivia
#[[What is the sequence of bacterial reverse transcriptase and can we clone that gene?]] Shamita
#[[Can we redesign the normal msDNA pathway to produce new segments of DNA of our choosing?]] All
#[[What are other available reverse transcriptases?]] Leland
#[[What other math problems (e.g. NP- complete) are accessible to us? Annie Siya Sun]]
#[[What is the relationship between 3-SAT and map coloring? Ashley Schnoor]]
#[[What activators are there that turn on a promoter without any help?]]
#[[Can we use protein interactions to compute? (Post-translation, proteases, quaternary structure) Will Vernon]]
#[[Could we do something with clocks/counting?]]
#[[Could we have/use multiple synthetic organelles in a cell?]]
#[[What ideas from previous iGEM teams are useful to us?]]
#[[Examples of Metabolic Pathways in E.coli]]

<center>'''Math-based'''</center>
#[[Can we get bacteria to solve a problem large enough to challenge a person?]]
#[[What interesting challenges or problems does origami offer?]]
#[[Can we produce a series of increasingly difficult goals that might be possible to produce in the lab?]]
#[[What has been done before and how can we improve upon that?]]
#[[We can perform some pilot experiments using synthesized DNA and later switch to msDNA (maybe).]]
#[[Can we address the Boolean Satisfiability (SAT) problem with a bacterial computer?]]
#[[How has 3SAT been addressed with a DNA computer? Can we use those methods?]]

#Can we get bacteria to solve a problem large enough to challenge a computer (probably not, but it is fun to think about)?
#[[What are some linear algebra applications for DNA origami?]]
#[[How can we use origami to solve 3-SAT problems?]]

<center>'''Behavior-based'''</center>
#[[What constructs are we testing?]]
#[[What school districts do we have access to?]]
#[[Where is the Synthetic Biology page we want high school teachers to use after the survey?]]
#[[Do you need any more input from the veterans before the survey is ready?]]

<center>'''Solving The 3SAT Problem Using Suppressor Logic'''</center>
#[[Can we solve a 3-SAT problem with supressor logic?]]

<center>'''Hin/Hix System'''</center>
Here is a link to the animation of the Hin/Hix system created by the "E. HOP living computer project folks.
[http://www.bio.davidson.edu/people/kahaynes/FAMU_talk/Living_computer.swf Hin/Hix Animation]

Can we solve a 3-SAT problem with supressor logic?

2009-05-27T14:57:44Z

Brs3298: /* Things we need to learn about */

Can we solve a 3-SAT problem with supressor logic?

2009-05-20T17:44:48Z

Brs3298: /* What is suppressor logic? */

== What is the 3-SAT problem? ==

''Advantages/Disadvantages to using the 3 SAT problem''

Advantages:

1) Seems to have an equal balance of mathematics and biology.

2) Eventually, there is promise to make the problem difficult enough whereas a computer would have a hard time solving it.

3) Introducing a new concept (supressor supressor logic)

Disadvantages:

1) Using the same concept/idea that has been used by previous Missouri Western/Davidson iGEM teams. (XOR gate)

2) Finding enough for everyone to work on for the entire summer.

3) Finding a promoter that is turned on without the use of a small particle.

== How did Sakamoto et al. use a DNA computer to solve a 3-SAT problem? ==

[http://gcat.davidson.edu/GcatWiki/images/3/39/Sakamoto_DNA_computer_3SAT.pdf Sakamoto et al.]

<center> [[Image:hairpin2.GIF]] </center>
Sakamoto used hairpin formations in single stranded DNA (ssDNA) as a molecular computer. Hairpin structures are formed when complimentary bases on the same strand attach to each other forming a loop. As shown in Picture B above, C and -C are compliments of each other, and bind together. Picture A is what a normal ssDNA should look like. Sakamoto used this self assembly of secondary structures on a satisfiability (SAT) problem. The problem had six inputs and ten clauses. An example of a clause would be (a or b or -c), and could include any combinations of inputs from a to f including -a to -f. If the problem is satisfied, the ssDNA stays in it's normal form. If the problem is not satisfied, the ssDNA forms a hairpin. Later in the paper it explains that the hairpin forming molecules can be removed from the others with certain techniques. 

The 10 clause 3-SAT problem solved in the paper is:

(a or b or –c) 
(a or c or d) 
(a or –c or –d) 
(-a or –c or d) 
(a or –c or e) 
(a or d or –f) 
(-a or c or d) 
(a or c or –d) 
(-a or –c or –d) 
(-a or c or –d) 

note: this problem uses inputs of a, b, c, d, e, -a, -c, -d, -f (f, -b, -e are not used)

== What is suppressor logic? ==

Will's Version of the PPT for SSL 3SAT
[http://gcat.davidson.edu/GcatWiki/images/6/6f/Will%27s_SSL_3SAT.ppt Will's Version of the PPT for SSL 3SAT]

Powerpoint on 3SAT SSL (Bryce and Clif)
[[3SAT]]

'''Suppressor Logic uses suppressor tRNAs as inputs to avoid frameshift mutations in the production of an output amino acid sequence'''

A frameshift is a genetic mutation caused by the addition or deletion of nucelotides to a given sequence which codes for a protein. Since codons are read in a series of three, the addition or deletion of nucleotides will disrupt the reading frame. This disruption will most likely cause the production of a nonfunctional protein.

<center> [[Image:suppressor4.GIF]] </center>

A frameshift occurs and, in this case, a guanine is added to the sequence. If nothing is done, enzyme A will not be made, meaning the clause will not be satisfied.

The suppressor tRNA allows the 4 letter sequence to be read as a single codon, therefore, keeping the protein on track. 

If more than one frameshift mutation is introduced into a coding sequence, then logical operators can be encoded. Suppressor a binds to CCCG, supressor b binds to CUGC, and suppressor c binds to ACCG below: 

# AUG CCCG CUG ... rest of Gene 
# AUG CCCG CUGC CUG ... rest of Gene 
# AUG CCCG gg CUGC AGG ... rest of Gene 
# AUG CCCG gg CUGC gg ACCG AGG ... rest of Gene 
 
{| align="center" border="1" cellpadding="8" cellspacing="0" style="background:lightcyan; font-weight:bold; text-align:center; width:45%"
|+
|- style="background:paleturquoise"
! style="width:15%" | Construct
! style="width:15%" | Gene
! style="width:15%" | Logical operation
|-
| 1 || Phenotype || a
|-
| 2 || Phenotype || a AND b
|-
| 3 || Phenotype || a OR b
|-
| 4 || Phenotype || a OR b OR c
|-
| 1 || Repressor|| NOT a
|-
| 2 || Repressor|| a NAND b
|-
| 3 || Repressor|| a NOR b
|-
| 4 || Repressor|| NOT (a OR b OR c)
|}

'''Logical clauses can be connected by AND operators if the proteins produced are part of a biochemical pathway.''' In this case, a AND b AND c: 

<center> [[Image:suppressor2.GIF]] </center>

== How could suppressor logic be used to solve the Sakamoto 3-SAT problem? ==

'''Definitions'''

Inputs = framshift suppressor tRNAs

Input value = supp a is 1, supp g is 0; supp b is 1, supp h is 0, etc. up to tth 6th pair of f and l

Logical clause (LC) = three inputs connected by OR, eg. (a OR b OR e)

LC using 4-base frameshift mutations: cag CCCG aa GGGC tt GTTG cag (may also have any multiple of 3 bases between the mutations) 

LC using 5-base frameshift mutations: cag CCCGC a GGGCG t GTTGC cag (may also have any multiple of 3 bases between the mutations) 

This makes an XOR logic gate as only one suppressor can be used at one time to maintain the reading frame.

Logical expression (LE) = string of LCs connected by AND 
The design below encodes LC1 AND LC2 AND LC3 

<center> [[Image:logical expression2.GIF]] </center>

'''Subroutine'''

1. Individual bacteral cells use Hin/hix system to randomly choose of of the 64 possible combinations of 6 inputs. Suppressors a and g represent 1 and 0 for the first input; suppressors b and h are 1 and 0 for the second input; etc. up to the sixth input with suppressors f and l (lower case L). The triangles are hix sites for Hin recombination. Whichever of the two suppressor tRNAs in an input pair is facing forward determines whether the value of that input is 1 or 0.

<center> [[Image:suppressor inputs.GIF]] </center>

2. Each bacterial cell carries out the following subroutine on each LC: IF LC=TRUE THEN "check the next LC" ELSEIF LC=FALSE "go get a new set of inputs with step 1" In order to do this, each of the activators below must turn on a repressor that turns off Hin production. Then if one of the activators is not made, Hin will be made, and new inputs will be established.

<center> [[Image:LE circuit.GIF]] </center>

3. If/when a bacterial cell finds a set of inputs that satisfies the entire LE (ie. a solution to the 3-SAT problem), it will glow green.

== Things we need to learn about ==

'''Note: We would need 12 different frameshift suppressor tRNAs to encode the Sakamoto 3-SAT problem'''

'''Discovery of frameshift tRNAs. How many are known?''' 

[http://gcat.davidson.edu/GcatWiki/images/5/59/MaglierySchultz2001JMB.pdf Magliery, Anderson, Schultz]

Library approach used to discover efficient suppressors of four-base codons AGGA, UAGA, CCCU, and CUAG using mutated versions of serine tRNA.

[http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf Anderson, Maglieri, Schultz]

Signals for translational bypassing (slipping and hopping): mRNA secondary structure, "hungry" (underused) codons, upstream Shine-Dalgarno-like (RBS) sequences 
Library approach extended in order to discover frameshift suppressor tRNAs with anticodons of size two to six bases. Two-base and six-base suppressors were not found, but the following five-base suppressors were found:

<center> [[Image:Table2_Anderson2002.GIF]] </center>

[http://gcat.davidson.edu/GcatWiki/images/a/ac/Dunham_2009_tRNA_structure.pdf Dunham et al.]

[http://gcat.davidson.edu/GcatWiki/images/c/c3/Hohsaka_2009_in_vitro_tln.pdf Hohsaka et al.]

'''Processing of tRNA precursors in E. coli''' 

[http://www.nature.com/embor/journal/v2/n1/full/embor501.html Mörl and Marchfelder] describe processing 

<center> [[Image:the_final_cut.GIF]] </center>

'''How many different frameshift suppressor tRNAs can be in one cell without causing toxicity?'''

Can we have twelve of them? If not, what is the maximum? Is there an impact of codon usage on this?

Can we have both 4- and 5-base suppressor tRNAs in one cell?

Are there particular combinations of suppressors that are more tolerated than others?

 
'''How many frameshift mutations can be suppressed in a single gene?'''

How close together can the frameshift mutations of a given LC be?

Can we mix 4- and 5-base frameshift mutations in a given LC?

Can we solve a 3-SAT problem with supressor logic?

2009-05-20T17:42:53Z

Brs3298: /* What is suppressor logic? */

== What is the 3-SAT problem? ==

''Advantages/Disadvantages to using the 3 SAT problem''

Advantages:

1) Seems to have an equal balance of mathematics and biology.

2) Eventually, there is promise to make the problem difficult enough whereas a computer would have a hard time solving it.

3) Introducing a new concept (supressor supressor logic)

Disadvantages:

1) Using the same concept/idea that has been used by previous Missouri Western/Davidson iGEM teams. (XOR gate)

2) Finding enough for everyone to work on for the entire summer.

3) Finding a promoter that is turned on without the use of a small particle.

== How did Sakamoto et al. use a DNA computer to solve a 3-SAT problem? ==

[http://gcat.davidson.edu/GcatWiki/images/3/39/Sakamoto_DNA_computer_3SAT.pdf Sakamoto et al.]

<center> [[Image:hairpin2.GIF]] </center>
Sakamoto used hairpin formations in single stranded DNA (ssDNA) as a molecular computer. Hairpin structures are formed when complimentary bases on the same strand attach to each other forming a loop. As shown in Picture B above, C and -C are compliments of each other, and bind together. Picture A is what a normal ssDNA should look like. Sakamoto used this self assembly of secondary structures on a satisfiability (SAT) problem. The problem had six inputs and ten clauses. An example of a clause would be (a or b or -c), and could include any combinations of inputs from a to f including -a to -f. If the problem is satisfied, the ssDNA stays in it's normal form. If the problem is not satisfied, the ssDNA forms a hairpin. Later in the paper it explains that the hairpin forming molecules can be removed from the others with certain techniques. 

The 10 clause 3-SAT problem solved in the paper is:

(a or b or –c) 
(a or c or d) 
(a or –c or –d) 
(-a or –c or d) 
(a or –c or e) 
(a or d or –f) 
(-a or c or d) 
(a or c or –d) 
(-a or –c or –d) 
(-a or c or –d) 

note: this problem uses inputs of a, b, c, d, e, -a, -c, -d, -f (f, -b, -e are not used)

== What is suppressor logic? ==

Will's Version of the PPT for SSL 3SAT
[http://gcat.davidson.edu/GcatWiki/images/6/6f/Will%27s_SSL_3SAT.ppt Will's Version of the PPT for SSL 3SAT]

Powerpoint on 3SAT SSL (Bryce and Clif)

'''Suppressor Logic uses suppressor tRNAs as inputs to avoid frameshift mutations in the production of an output amino acid sequence'''

A frameshift is a genetic mutation caused by the addition or deletion of nucelotides to a given sequence which codes for a protein. Since codons are read in a series of three, the addition or deletion of nucleotides will disrupt the reading frame. This disruption will most likely cause the production of a nonfunctional protein.

<center> [[Image:suppressor4.GIF]] </center>

A frameshift occurs and, in this case, a guanine is added to the sequence. If nothing is done, enzyme A will not be made, meaning the clause will not be satisfied.

The suppressor tRNA allows the 4 letter sequence to be read as a single codon, therefore, keeping the protein on track. 

If more than one frameshift mutation is introduced into a coding sequence, then logical operators can be encoded. Suppressor a binds to CCCG, supressor b binds to CUGC, and suppressor c binds to ACCG below: 

# AUG CCCG CUG ... rest of Gene 
# AUG CCCG CUGC CUG ... rest of Gene 
# AUG CCCG gg CUGC AGG ... rest of Gene 
# AUG CCCG gg CUGC gg ACCG AGG ... rest of Gene 
 
{| align="center" border="1" cellpadding="8" cellspacing="0" style="background:lightcyan; font-weight:bold; text-align:center; width:45%"
|+
|- style="background:paleturquoise"
! style="width:15%" | Construct
! style="width:15%" | Gene
! style="width:15%" | Logical operation
|-
| 1 || Phenotype || a
|-
| 2 || Phenotype || a AND b
|-
| 3 || Phenotype || a OR b
|-
| 4 || Phenotype || a OR b OR c
|-
| 1 || Repressor|| NOT a
|-
| 2 || Repressor|| a NAND b
|-
| 3 || Repressor|| a NOR b
|-
| 4 || Repressor|| NOT (a OR b OR c)
|}

'''Logical clauses can be connected by AND operators if the proteins produced are part of a biochemical pathway.''' In this case, a AND b AND c: 

<center> [[Image:suppressor2.GIF]] </center>

== How could suppressor logic be used to solve the Sakamoto 3-SAT problem? ==

'''Definitions'''

Inputs = framshift suppressor tRNAs

Input value = supp a is 1, supp g is 0; supp b is 1, supp h is 0, etc. up to tth 6th pair of f and l

Logical clause (LC) = three inputs connected by OR, eg. (a OR b OR e)

LC using 4-base frameshift mutations: cag CCCG aa GGGC tt GTTG cag (may also have any multiple of 3 bases between the mutations) 

LC using 5-base frameshift mutations: cag CCCGC a GGGCG t GTTGC cag (may also have any multiple of 3 bases between the mutations) 

This makes an XOR logic gate as only one suppressor can be used at one time to maintain the reading frame.

Logical expression (LE) = string of LCs connected by AND 
The design below encodes LC1 AND LC2 AND LC3 

<center> [[Image:logical expression2.GIF]] </center>

'''Subroutine'''

1. Individual bacteral cells use Hin/hix system to randomly choose of of the 64 possible combinations of 6 inputs. Suppressors a and g represent 1 and 0 for the first input; suppressors b and h are 1 and 0 for the second input; etc. up to the sixth input with suppressors f and l (lower case L). The triangles are hix sites for Hin recombination. Whichever of the two suppressor tRNAs in an input pair is facing forward determines whether the value of that input is 1 or 0.

<center> [[Image:suppressor inputs.GIF]] </center>

2. Each bacterial cell carries out the following subroutine on each LC: IF LC=TRUE THEN "check the next LC" ELSEIF LC=FALSE "go get a new set of inputs with step 1" In order to do this, each of the activators below must turn on a repressor that turns off Hin production. Then if one of the activators is not made, Hin will be made, and new inputs will be established.

<center> [[Image:LE circuit.GIF]] </center>

3. If/when a bacterial cell finds a set of inputs that satisfies the entire LE (ie. a solution to the 3-SAT problem), it will glow green.

== Things we need to learn about ==

'''Note: We would need 12 different frameshift suppressor tRNAs to encode the Sakamoto 3-SAT problem'''

'''Discovery of frameshift tRNAs. How many are known?''' 

[http://gcat.davidson.edu/GcatWiki/images/5/59/MaglierySchultz2001JMB.pdf Magliery, Anderson, Schultz]

Library approach used to discover efficient suppressors of four-base codons AGGA, UAGA, CCCU, and CUAG using mutated versions of serine tRNA.

[http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf Anderson, Maglieri, Schultz]

Signals for translational bypassing (slipping and hopping): mRNA secondary structure, "hungry" (underused) codons, upstream Shine-Dalgarno-like (RBS) sequences 
Library approach extended in order to discover frameshift suppressor tRNAs with anticodons of size two to six bases. Two-base and six-base suppressors were not found, but the following five-base suppressors were found:

<center> [[Image:Table2_Anderson2002.GIF]] </center>

[http://gcat.davidson.edu/GcatWiki/images/a/ac/Dunham_2009_tRNA_structure.pdf Dunham et al.]

[http://gcat.davidson.edu/GcatWiki/images/c/c3/Hohsaka_2009_in_vitro_tln.pdf Hohsaka et al.]

'''Processing of tRNA precursors in E. coli''' 

[http://www.nature.com/embor/journal/v2/n1/full/embor501.html Mörl and Marchfelder] describe processing 

<center> [[Image:the_final_cut.GIF]] </center>

'''How many different frameshift suppressor tRNAs can be in one cell without causing toxicity?'''

Can we have twelve of them? If not, what is the maximum? Is there an impact of codon usage on this?

Can we have both 4- and 5-base suppressor tRNAs in one cell?

Are there particular combinations of suppressors that are more tolerated than others?

 
'''How many frameshift mutations can be suppressed in a single gene?'''

How close together can the frameshift mutations of a given LC be?

Can we mix 4- and 5-base frameshift mutations in a given LC?

Missouri Western/Davidson iGEM2009

2009-05-14T13:48:07Z

Brs3298:

This space will be used starting April, 2009 for brainstorming and a shared whiteboard space.

[http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Davidson_Protocols Davidson Lab Protocols] 
[http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/MWSU_protocols MWSU Lab Protocols] 
[http://gcat.davidson.edu/sybr-u/bmc.html BioMath Connections Page] 
[http://gcat.davidson.edu/GCATalog-r2.1/GCATalog.htm GCAT-alog Freezer Stocks] 

We need to learn more about these topics:
<center>'''Biology-based'''</center>
#[[What is msDNA?]]
#[[How is msDNA normally produced?]] Olivia/Alyndria
#[[How is msDNA stored in E. coli?]] Olivia
#[[How many copies are carried per cell?]] Alyndria
#[[What is the sequence of bacterial reverse transcriptase and can we clone that gene?]] Shamita
#[[Can we redesign the normal msDNA pathway to produce new segments of DNA of our choosing?]] All
#[[Can we use suppressor tRNAs to encode logical operators (suppressor suppressor logic, SSL)?]]
#[[What are other available reverse transcriptases?]] Leland
#[[Can we solve a 3-SAT problem with supressor logic?]]
#[[What role can physical modeling of protein structure play in our project?]] Romina
#[[What other math problems (e.g. NP- complete) are accessible to us? Siya Sun]]
#[[What is the relationship between 3-SAT and map coloring? Ashley Schnoor]]
#[[What role can physical modeling of proteins play in our project? Eric Sawyer]]
#[[What activators are there that turn on a promoter without any help?]]
#[[What other cool reporters are there? (Discrete On/Off or Continuous) Bryce Szczepanik]]
#[[Can we use promoter strength/opposite directions to subtract? Clif Davis]]
#[[Can we use protein interactions to compute? (Post-translation, proteases, quaternary structure) Will Vernon]]
#[[Could we do something with clocks/counting?]]
#[[Could we have/use multiple synthetic organelles in a cell?]]
#[[What ideas from previous iGEM teams are useful to us?]]

<center>'''Math-based'''</center>
#What interesting challenges or problems does origami offer?
#Can we produce a series of increasingly difficult goals that might be possible to produce in the lab?
#What has been done before and how can we improve upon that?
#We can perform some pilot experiments using synthesized DNA and later switch to msDNA (maybe).
#Can we address the Boolean Satisfiability (SAT) problem with a bacterial computer?
#How has 3SAT been addressed with a DNA computer? Can we use those methods?
#[[Can we get bacteria to solve a problem large enough to challenge a person?]]
#Can we get bacteria to solve a problem large enough to challenge a computer (probably not, but it is fun to think about)?
#What are some linear algebra applications for DNA origami?
#How can we use origami to solve 3-SAT problems?

<center>'''Behavior-based'''</center>
#[[What constructs are we testing?]]
#[[What school districts do we have access to?]]
#[[Where is the Synthetic Biology page we want high school teachers to use after the survey?]]
#[[Do you need any more input from the veterans before the survey is ready?]]

Can we solve a 3-SAT problem with supressor logic?

2009-05-12T15:34:14Z

Brs3298: /* What is the 3-SAT problem? */

== What is the 3-SAT problem? ==

''Advantages/Disadvantages to using the 3 SAT problem''

Advantages:

1) Seems to have an equal balance of mathematics and biology.

2) Eventually, there is promise to make the problem difficult enough whereas a computer would have a hard time solving it.

3) Introducing a new concept (supressor supressor logic)

Disadvantages:

1) Using the same concept/idea that has been used by previous Missouri Western/Davidson iGEM teams. (XOR gate)

2) Finding enough for everyone to work on for the entire summer.

3) Finding a promoter that is turned on without the use of a small particle.

== How did Sakamoto et al. use a DNA computer to solve a 3-SAT problem? ==

[http://gcat.davidson.edu/GcatWiki/images/3/39/Sakamoto_DNA_computer_3SAT.pdf Sakamoto et al.]

<center> [[Image:hairpin2.GIF]] </center>
Sakamoto used hairpin formations in single stranded DNA (ssDNA) as a molecular computer. Hairpin structures are formed when complimentary bases on the same strand attach to each other forming a loop. As shown in Picture B above, C and -C are compliments of each other, and bind together. Picture A is what a normal ssDNA should look like. Sakamoto used this self assembly of secondary structures on a satisfiability (SAT) problem. The problem had six inputs and ten clauses. An example of a clause would be (a or b or -c), and could include any combinations of inputs from a to f including -a to -f. If the problem is satisfied, the ssDNA stays in it's normal form. If the problem is not satisfied, the ssDNA forms a hairpin. Later in the paper it explains that the hairpin forming molecules can be removed from the others with certain techniques. 

The 10 clause 3-SAT problem solved in the paper is:

(a or b or –c) 
(a or c or d) 
(a or –c or –d) 
(-a or –c or d) 
(a or –c or e) 
(a or d or –f) 
(-a or c or d) 
(a or c or –d) 
(-a or –c or –d) 
(-a or c or –d) 

note: this problem uses inputs of a, b, c, d, e, -a, -c, -d, -f (f, -b, -e are not used)

== What is suppressor logic? ==

'''Suppressor Logic uses suppressor tRNAs as inputs to avoid frameshift mutations in the production of an output amino acid sequence'''

A frameshift is a genetic mutation caused by the addition or deletion of nucelotides to a given sequence which codes for a protein. Since codons are read in a series of three, the addition or deletion of nucleotides will disrupt the reading frame. This disruption will most likely cause the production of a nonfunctional protein.

<center> [[Image:suppressor4.GIF]] </center>

A frameshift occurs and, in this case, a guanine is added to the sequence. If nothing is done, enzyme A will not be made, meaning the clause will not be satisfied.

The suppressor tRNA allows the 4 letter sequence to be read as a single codon, therefore, keeping the protein on track. 

If more than one frameshift mutation is introduced into a coding sequence, then logical operators can be encoded. Suppressor a binds to CCCG, supressor b binds to CUGC, and suppressor c binds to ACCG below: 

# AUG CCCG CUG ... rest of Gene 
# AUG CCCG CUGC CUG ... rest of Gene 
# AUG CCCG gg CUGC AGG ... rest of Gene 
# AUG CCCG gg CUGC gg ACCG AGG ... rest of Gene 
 
{| align="center" border="1" cellpadding="8" cellspacing="0" style="background:lightcyan; font-weight:bold; text-align:center; width:45%"
|+
|- style="background:paleturquoise"
! style="width:15%" | Construct
! style="width:15%" | Gene
! style="width:15%" | Logical operation
|-
| 1 || Phenotype || a
|-
| 2 || Phenotype || a AND b
|-
| 3 || Phenotype || a OR b
|-
| 4 || Phenotype || a OR b OR c
|-
| 1 || Repressor|| NOT a
|-
| 2 || Repressor|| a NAND b
|-
| 3 || Repressor|| a NOR b
|-
| 4 || Repressor|| NOT (a OR b OR c)
|}

'''Logical clauses can be connected by AND operators if the proteins produced are part of a biochemical pathway.''' In this case, a AND b AND c: 

<center> [[Image:suppressor2.GIF]] </center>

== How could suppressor logic be used to solve the Sakamoto 3-SAT problem? ==

'''Definitions'''

Inputs = framshift suppressor tRNAs

Input value = supp a is 1, supp g is 0; supp b is 1, supp h is 0, etc. up to tth 6th pair of f and l

Logical clause (LC) = three inputs connected by OR, eg. (a OR b OR e)

LC using 4-base frameshift mutations: cag CCCG aa GGGC tt GTTG cag (may also have any multiple of 3 bases between the mutations) 

LC using 5-base frameshift mutations: cag CCCGC a GGGCG t GTTGC cag (may also have any multiple of 3 bases between the mutations) 

This makes an XOR logic gate as only one suppressor can be used at one time to maintain the reading frame.

Logical expression (LE) = string of LCs connected by AND 
The design below encodes LC1 AND LC2 AND LC3 

<center> [[Image:logical expression2.GIF]] </center>

'''Subroutine'''

1. Individual bacteral cells use Hin/hix system to randomly choose of of the 64 possible combinations of 6 inputs. Suppressors a and g represent 1 and 0 for the first input; suppressors b and h are 1 and 0 for the second input; etc. up to the sixth input with suppressors f and l (lower case L). The triangles are hix sites for Hin recombination. Whichever of the two suppressor tRNAs in an input pair is facing forward determines whether the value of that input is 1 or 0.

<center> [[Image:suppressor inputs.GIF]] </center>

2. Each bacterial cell carries out the following subroutine on each LC: IF LC=TRUE THEN "check the next LC" ELSEIF LC=FALSE "go get a new set of inputs with step 1" In order to do this, each of the activators below must turn on a repressor that turns off Hin production. Then if one of the activators is not made, Hin will be made, and new inputs will be established.

<center> [[Image:LE circuit.GIF]] </center>

3. If/when a bacterial cell finds a set of inputs that satisfies the entire LE (ie. a solution to the 3-SAT problem), it will glow green.

== Things we need to learn about ==

'''Note: We would need 12 different frameshift suppressor tRNAs to encode the Sakamoto 3-SAT problem'''

'''Discovery of frameshift tRNAs. How many are known?''' 

[http://gcat.davidson.edu/GcatWiki/images/5/59/MaglierySchultz2001JMB.pdf Magliery, Anderson, Schultz]

Library approach used to discover efficient suppressors of four-base codons AGGA, UAGA, CCCU, and CUAG using mutated versions of serine tRNA.

[http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf Anderson, Maglieri, Schultz]

Signals for translational bypassing (slipping and hopping): mRNA secondary structure, "hungry" (underused) codons, upstream Shine-Dalgarno-like (RBS) sequences 
Library approach extended in order to discover frameshift suppressor tRNAs with anticodons of size two to six bases. Two-base and six-base suppressors were not found, but the following five-base suppressors were found:

<center> [[Image:Table2_Anderson2002.GIF]] </center>

[http://gcat.davidson.edu/GcatWiki/images/a/ac/Dunham_2009_tRNA_structure.pdf Dunham et al.]

[http://gcat.davidson.edu/GcatWiki/images/c/c3/Hohsaka_2009_in_vitro_tln.pdf Hohsaka et al.]

'''Processing of tRNA precursors in E. coli''' 

[http://www.nature.com/embor/journal/v2/n1/full/embor501.html Mörl and Marchfelder] describe processing 

<center> [[Image:the_final_cut.GIF]] </center>

'''How many different frameshift suppressor tRNAs can be in one cell without causing toxicity?'''

Can we have twelve of them? If not, what is the maximum? Is there an impact of codon usage on this?

Can we have both 4- and 5-base suppressor tRNAs in one cell?

Are there particular combinations of suppressors that are more tolerated than others?

 
'''How many frameshift mutations can be suppressed in a single gene?'''

How close together can the frameshift mutations of a given LC be?

Can we mix 4- and 5-base frameshift mutations in a given LC?

Can we solve a 3-SAT problem with supressor logic?

2009-05-12T15:24:56Z

Brs3298: /* What is the 3-SAT problem? */

== What is the 3-SAT problem? ==

''Advantages/Disadvantages to using the 3 SAT problem''

Advantages:

1) Seems to have an equal balance of mathematics and biology.

2) Eventually, there is promise to make the problem difficult enough whereas a computer would have a hard time solving it.

3) Introducing a new concept (supressor supressor logic)

Disadvantages:

1)

== How did Sakamoto et al. use a DNA computer to solve a 3-SAT problem? ==

[http://gcat.davidson.edu/GcatWiki/images/3/39/Sakamoto_DNA_computer_3SAT.pdf Sakamoto et al.]

<center> [[Image:hairpin2.GIF]] </center>
Sakamoto used hairpin formations in single stranded DNA (ssDNA) as a molecular computer. Hairpin structures are formed when complimentary bases on the same strand attach to each other forming a loop. As shown in Picture B above, C and -C are compliments of each other, and bind together. Picture A is what a normal ssDNA should look like. Sakamoto used this self assembly of secondary structures on a satisfiability (SAT) problem. The problem had six inputs and ten clauses. An example of a clause would be (a or b or -c), and could include any combinations of inputs from a to f including -a to -f. If the problem is satisfied, the ssDNA stays in it's normal form. If the problem is not satisfied, the ssDNA forms a hairpin. Later in the paper it explains that the hairpin forming molecules can be removed from the others with certain techniques. 

The 10 clause 3-SAT problem solved in the paper is:

(a or b or –c) 
(a or c or d) 
(a or –c or –d) 
(-a or –c or d) 
(a or –c or e) 
(a or d or –f) 
(-a or c or d) 
(a or c or –d) 
(-a or –c or –d) 
(-a or c or –d) 

note: this problem uses inputs of a, b, c, d, e, -a, -c, -d, -f (f, -b, -e are not used)

== What is suppressor logic? ==

'''Suppressor Logic uses suppressor tRNAs as inputs to avoid frameshift mutations in the production of an output amino acid sequence'''

A frameshift is a genetic mutation caused by the addition or deletion of nucelotides to a given sequence which codes for a protein. Since codons are read in a series of three, the addition or deletion of nucleotides will disrupt the reading frame. This disruption will most likely cause the production of a nonfunctional protein.

<center> [[Image:suppressor4.GIF]] </center>

A frameshift occurs and, in this case, a guanine is added to the sequence. If nothing is done, enzyme A will not be made, meaning the clause will not be satisfied.

The suppressor tRNA allows the 4 letter sequence to be read as a single codon, therefore, keeping the protein on track. 

If more than one frameshift mutation is introduced into a coding sequence, then logical operators can be encoded. Suppressor a binds to CCCG, supressor b binds to CUGC, and suppressor c binds to ACCG below: 

# AUG CCCG CUG ... rest of Gene 
# AUG CCCG CUGC CUG ... rest of Gene 
# AUG CCCG gg CUGC AGG ... rest of Gene 
# AUG CCCG gg CUGC gg ACCG AGG ... rest of Gene 
 
{| align="center" border="1" cellpadding="8" cellspacing="0" style="background:lightcyan; font-weight:bold; text-align:center; width:45%"
|+
|- style="background:paleturquoise"
! style="width:15%" | Construct
! style="width:15%" | Gene
! style="width:15%" | Logical operation
|-
| 1 || Phenotype || a
|-
| 2 || Phenotype || a AND b
|-
| 3 || Phenotype || a OR b
|-
| 4 || Phenotype || a OR b OR c
|-
| 1 || Repressor|| NOT a
|-
| 2 || Repressor|| a NAND b
|-
| 3 || Repressor|| a NOR b
|-
| 4 || Repressor|| NOT (a OR b OR c)
|}

'''Logical clauses can be connected by AND operators if the proteins produced are part of a biochemical pathway.''' In this case, a AND b AND c: 

<center> [[Image:suppressor2.GIF]] </center>

== How could suppressor logic be used to solve the Sakamoto 3-SAT problem? ==

'''Definitions'''

Inputs = framshift suppressor tRNAs

Input value = supp a is 1, supp g is 0; supp b is 1, supp h is 0, etc. up to tth 6th pair of f and l

Logical clause (LC) = three inputs connected by OR, eg. (a OR b OR e)

LC using 4-base frameshift mutations: cag CCCG aa GGGC tt GTTG cag (may also have any multiple of 3 bases between the mutations) 

LC using 5-base frameshift mutations: cag CCCGC a GGGCG t GTTGC cag (may also have any multiple of 3 bases between the mutations) 

This makes an XOR logic gate as only one suppressor can be used at one time to maintain the reading frame.

Logical expression (LE) = string of LCs connected by AND 
The design below encodes LC1 AND LC2 AND LC3 

<center> [[Image:logical expression2.GIF]] </center>

'''Subroutine'''

1. Individual bacteral cells use Hin/hix system to randomly choose of of the 64 possible combinations of 6 inputs. Suppressors a and g represent 1 and 0 for the first input; suppressors b and h are 1 and 0 for the second input; etc. up to the sixth input with suppressors f and l (lower case L). The triangles are hix sites for Hin recombination. Whichever of the two suppressor tRNAs in an input pair is facing forward determines whether the value of that input is 1 or 0.

<center> [[Image:suppressor inputs.GIF]] </center>

2. Each bacterial cell carries out the following subroutine on each LC: IF LC=TRUE THEN "check the next LC" ELSEIF LC=FALSE "go get a new set of inputs with step 1" In order to do this, each of the activators below must turn on a repressor that turns off Hin production. Then if one of the activators is not made, Hin will be made, and new inputs will be established.

<center> [[Image:LE circuit.GIF]] </center>

3. If/when a bacterial cell finds a set of inputs that satisfies the entire LE (ie. a solution to the 3-SAT problem), it will glow green.

== Things we need to learn about ==

'''Note: We would need 12 different frameshift suppressor tRNAs to encode the Sakamoto 3-SAT problem'''

'''Discovery of frameshift tRNAs. How many are known?''' 

[http://gcat.davidson.edu/GcatWiki/images/5/59/MaglierySchultz2001JMB.pdf Magliery, Anderson, Schultz]

Library approach used to discover efficient suppressors of four-base codons AGGA, UAGA, CCCU, and CUAG using mutated versions of serine tRNA.

[http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf Anderson, Maglieri, Schultz]

Signals for translational bypassing (slipping and hopping): mRNA secondary structure, "hungry" (underused) codons, upstream Shine-Dalgarno-like (RBS) sequences 
Library approach extended in order to discover frameshift suppressor tRNAs with anticodons of size two to six bases. Two-base and six-base suppressors were not found, but the following five-base suppressors were found:

<center> [[Image:Table2_Anderson2002.GIF]] </center>

[http://gcat.davidson.edu/GcatWiki/images/a/ac/Dunham_2009_tRNA_structure.pdf Dunham et al.]

[http://gcat.davidson.edu/GcatWiki/images/c/c3/Hohsaka_2009_in_vitro_tln.pdf Hohsaka et al.]

'''Processing of tRNA precursors in E. coli''' 

[http://www.nature.com/embor/journal/v2/n1/full/embor501.html Mörl and Marchfelder] describe processing 

<center> [[Image:the_final_cut.GIF]] </center>

'''How many different frameshift suppressor tRNAs can be in one cell without causing toxicity?'''

Can we have twelve of them? If not, what is the maximum? Is there an impact of codon usage on this?

Can we have both 4- and 5-base suppressor tRNAs in one cell?

Are there particular combinations of suppressors that are more tolerated than others?

 
'''How many frameshift mutations can be suppressed in a single gene?'''

How close together can the frameshift mutations of a given LC be?

Can we mix 4- and 5-base frameshift mutations in a given LC?

Can we solve a 3-SAT problem with supressor logic?

2009-05-12T15:23:04Z

Brs3298: /* What is the 3-SAT problem? */

== What is the 3-SAT problem? ==

''Advantages/Disadvantages to using the 3 SAT problem''

Advantages:

1) Seems to have an equal balance of mathematics and biology.

2) Eventually, there is promise to make the problem difficult enough whereas a computer would have a hard time solving it.

3) Introducing a new concept (supressor supressor logic)

== How did Sakamoto et al. use a DNA computer to solve a 3-SAT problem? ==

[http://gcat.davidson.edu/GcatWiki/images/3/39/Sakamoto_DNA_computer_3SAT.pdf Sakamoto et al.]

<center> [[Image:hairpin2.GIF]] </center>
Sakamoto used hairpin formations in single stranded DNA (ssDNA) as a molecular computer. Hairpin structures are formed when complimentary bases on the same strand attach to each other forming a loop. As shown in Picture B above, C and -C are compliments of each other, and bind together. Picture A is what a normal ssDNA should look like. Sakamoto used this self assembly of secondary structures on a satisfiability (SAT) problem. The problem had six inputs and ten clauses. An example of a clause would be (a or b or -c), and could include any combinations of inputs from a to f including -a to -f. If the problem is satisfied, the ssDNA stays in it's normal form. If the problem is not satisfied, the ssDNA forms a hairpin. Later in the paper it explains that the hairpin forming molecules can be removed from the others with certain techniques. 

The 10 clause 3-SAT problem solved in the paper is:

(a or b or –c) 
(a or c or d) 
(a or –c or –d) 
(-a or –c or d) 
(a or –c or e) 
(a or d or –f) 
(-a or c or d) 
(a or c or –d) 
(-a or –c or –d) 
(-a or c or –d) 

note: this problem uses inputs of a, b, c, d, e, -a, -c, -d, -f (f, -b, -e are not used)

== What is suppressor logic? ==

'''Suppressor Logic uses suppressor tRNAs as inputs to avoid frameshift mutations in the production of an output amino acid sequence'''

A frameshift is a genetic mutation caused by the addition or deletion of nucelotides to a given sequence which codes for a protein. Since codons are read in a series of three, the addition or deletion of nucleotides will disrupt the reading frame. This disruption will most likely cause the production of a nonfunctional protein.

<center> [[Image:suppressor4.GIF]] </center>

A frameshift occurs and, in this case, a guanine is added to the sequence. If nothing is done, enzyme A will not be made, meaning the clause will not be satisfied.

The suppressor tRNA allows the 4 letter sequence to be read as a single codon, therefore, keeping the protein on track. 

If more than one frameshift mutation is introduced into a coding sequence, then logical operators can be encoded. Suppressor a binds to CCCG, supressor b binds to CUGC, and suppressor c binds to ACCG below: 

# AUG CCCG CUG ... rest of Gene 
# AUG CCCG CUGC CUG ... rest of Gene 
# AUG CCCG gg CUGC AGG ... rest of Gene 
# AUG CCCG gg CUGC gg ACCG AGG ... rest of Gene 
 
{| align="center" border="1" cellpadding="8" cellspacing="0" style="background:lightcyan; font-weight:bold; text-align:center; width:45%"
|+
|- style="background:paleturquoise"
! style="width:15%" | Construct
! style="width:15%" | Gene
! style="width:15%" | Logical operation
|-
| 1 || Phenotype || a
|-
| 2 || Phenotype || a AND b
|-
| 3 || Phenotype || a OR b
|-
| 4 || Phenotype || a OR b OR c
|-
| 1 || Repressor|| NOT a
|-
| 2 || Repressor|| a NAND b
|-
| 3 || Repressor|| a NOR b
|-
| 4 || Repressor|| NOT (a OR b OR c)
|}

'''Logical clauses can be connected by AND operators if the proteins produced are part of a biochemical pathway.''' In this case, a AND b AND c: 

<center> [[Image:suppressor2.GIF]] </center>

== How could suppressor logic be used to solve the Sakamoto 3-SAT problem? ==

'''Definitions'''

Inputs = framshift suppressor tRNAs

Input value = supp a is 1, supp g is 0; supp b is 1, supp h is 0, etc. up to tth 6th pair of f and l

Logical clause (LC) = three inputs connected by OR, eg. (a OR b OR e)

LC using 4-base frameshift mutations: cag CCCG aa GGGC tt GTTG cag (may also have any multiple of 3 bases between the mutations) 

LC using 5-base frameshift mutations: cag CCCGC a GGGCG t GTTGC cag (may also have any multiple of 3 bases between the mutations) 

This makes an XOR logic gate as only one suppressor can be used at one time to maintain the reading frame.

Logical expression (LE) = string of LCs connected by AND 
The design below encodes LC1 AND LC2 AND LC3 

<center> [[Image:logical expression2.GIF]] </center>

'''Subroutine'''

1. Individual bacteral cells use Hin/hix system to randomly choose of of the 64 possible combinations of 6 inputs. Suppressors a and g represent 1 and 0 for the first input; suppressors b and h are 1 and 0 for the second input; etc. up to the sixth input with suppressors f and l (lower case L). The triangles are hix sites for Hin recombination. Whichever of the two suppressor tRNAs in an input pair is facing forward determines whether the value of that input is 1 or 0.

<center> [[Image:suppressor inputs.GIF]] </center>

2. Each bacterial cell carries out the following subroutine on each LC: IF LC=TRUE THEN "check the next LC" ELSEIF LC=FALSE "go get a new set of inputs with step 1" In order to do this, each of the activators below must turn on a repressor that turns off Hin production. Then if one of the activators is not made, Hin will be made, and new inputs will be established.

<center> [[Image:LE circuit.GIF]] </center>

3. If/when a bacterial cell finds a set of inputs that satisfies the entire LE (ie. a solution to the 3-SAT problem), it will glow green.

== Things we need to learn about ==

'''Note: We would need 12 different frameshift suppressor tRNAs to encode the Sakamoto 3-SAT problem'''

'''Discovery of frameshift tRNAs. How many are known?''' 

[http://gcat.davidson.edu/GcatWiki/images/5/59/MaglierySchultz2001JMB.pdf Magliery, Anderson, Schultz]

Library approach used to discover efficient suppressors of four-base codons AGGA, UAGA, CCCU, and CUAG using mutated versions of serine tRNA.

[http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf Anderson, Maglieri, Schultz]

Signals for translational bypassing (slipping and hopping): mRNA secondary structure, "hungry" (underused) codons, upstream Shine-Dalgarno-like (RBS) sequences 
Library approach extended in order to discover frameshift suppressor tRNAs with anticodons of size two to six bases. Two-base and six-base suppressors were not found, but the following five-base suppressors were found:

<center> [[Image:Table2_Anderson2002.GIF]] </center>

[http://gcat.davidson.edu/GcatWiki/images/a/ac/Dunham_2009_tRNA_structure.pdf Dunham et al.]

[http://gcat.davidson.edu/GcatWiki/images/c/c3/Hohsaka_2009_in_vitro_tln.pdf Hohsaka et al.]

'''Processing of tRNA precursors in E. coli''' 

[http://www.nature.com/embor/journal/v2/n1/full/embor501.html Mörl and Marchfelder] describe processing 

<center> [[Image:the_final_cut.GIF]] </center>

'''How many different frameshift suppressor tRNAs can be in one cell without causing toxicity?'''

Can we have twelve of them? If not, what is the maximum? Is there an impact of codon usage on this?

Can we have both 4- and 5-base suppressor tRNAs in one cell?

Are there particular combinations of suppressors that are more tolerated than others?

 
'''How many frameshift mutations can be suppressed in a single gene?'''

How close together can the frameshift mutations of a given LC be?

Can we mix 4- and 5-base frameshift mutations in a given LC?

Can we solve a 3-SAT problem with supressor logic?

2009-05-12T15:22:47Z

Brs3298: /* What is the 3-SAT problem? */

== What is the 3-SAT problem? ==

''Advantages/Disadvantages to using the 3 SAT problem''

Advantages:
1) Seems to have an equal balance of mathematics and biology.
2) Eventually, there is promise to make the problem difficult enough whereas a computer would have a hard time solving it.
3) Introducing a new concept (supressor supressor logic)

== How did Sakamoto et al. use a DNA computer to solve a 3-SAT problem? ==

[http://gcat.davidson.edu/GcatWiki/images/3/39/Sakamoto_DNA_computer_3SAT.pdf Sakamoto et al.]

<center> [[Image:hairpin2.GIF]] </center>
Sakamoto used hairpin formations in single stranded DNA (ssDNA) as a molecular computer. Hairpin structures are formed when complimentary bases on the same strand attach to each other forming a loop. As shown in Picture B above, C and -C are compliments of each other, and bind together. Picture A is what a normal ssDNA should look like. Sakamoto used this self assembly of secondary structures on a satisfiability (SAT) problem. The problem had six inputs and ten clauses. An example of a clause would be (a or b or -c), and could include any combinations of inputs from a to f including -a to -f. If the problem is satisfied, the ssDNA stays in it's normal form. If the problem is not satisfied, the ssDNA forms a hairpin. Later in the paper it explains that the hairpin forming molecules can be removed from the others with certain techniques. 

The 10 clause 3-SAT problem solved in the paper is:

(a or b or –c) 
(a or c or d) 
(a or –c or –d) 
(-a or –c or d) 
(a or –c or e) 
(a or d or –f) 
(-a or c or d) 
(a or c or –d) 
(-a or –c or –d) 
(-a or c or –d) 

note: this problem uses inputs of a, b, c, d, e, -a, -c, -d, -f (f, -b, -e are not used)

== What is suppressor logic? ==

'''Suppressor Logic uses suppressor tRNAs as inputs to avoid frameshift mutations in the production of an output amino acid sequence'''

A frameshift is a genetic mutation caused by the addition or deletion of nucelotides to a given sequence which codes for a protein. Since codons are read in a series of three, the addition or deletion of nucleotides will disrupt the reading frame. This disruption will most likely cause the production of a nonfunctional protein.

<center> [[Image:suppressor4.GIF]] </center>

A frameshift occurs and, in this case, a guanine is added to the sequence. If nothing is done, enzyme A will not be made, meaning the clause will not be satisfied.

The suppressor tRNA allows the 4 letter sequence to be read as a single codon, therefore, keeping the protein on track. 

If more than one frameshift mutation is introduced into a coding sequence, then logical operators can be encoded. Suppressor a binds to CCCG, supressor b binds to CUGC, and suppressor c binds to ACCG below: 

# AUG CCCG CUG ... rest of Gene 
# AUG CCCG CUGC CUG ... rest of Gene 
# AUG CCCG gg CUGC AGG ... rest of Gene 
# AUG CCCG gg CUGC gg ACCG AGG ... rest of Gene 
 
{| align="center" border="1" cellpadding="8" cellspacing="0" style="background:lightcyan; font-weight:bold; text-align:center; width:45%"
|+
|- style="background:paleturquoise"
! style="width:15%" | Construct
! style="width:15%" | Gene
! style="width:15%" | Logical operation
|-
| 1 || Phenotype || a
|-
| 2 || Phenotype || a AND b
|-
| 3 || Phenotype || a OR b
|-
| 4 || Phenotype || a OR b OR c
|-
| 1 || Repressor|| NOT a
|-
| 2 || Repressor|| a NAND b
|-
| 3 || Repressor|| a NOR b
|-
| 4 || Repressor|| NOT (a OR b OR c)
|}

'''Logical clauses can be connected by AND operators if the proteins produced are part of a biochemical pathway.''' In this case, a AND b AND c: 

<center> [[Image:suppressor2.GIF]] </center>

== How could suppressor logic be used to solve the Sakamoto 3-SAT problem? ==

'''Definitions'''

Inputs = framshift suppressor tRNAs

Input value = supp a is 1, supp g is 0; supp b is 1, supp h is 0, etc. up to tth 6th pair of f and l

Logical clause (LC) = three inputs connected by OR, eg. (a OR b OR e)

LC using 4-base frameshift mutations: cag CCCG aa GGGC tt GTTG cag (may also have any multiple of 3 bases between the mutations) 

LC using 5-base frameshift mutations: cag CCCGC a GGGCG t GTTGC cag (may also have any multiple of 3 bases between the mutations) 

This makes an XOR logic gate as only one suppressor can be used at one time to maintain the reading frame.

Logical expression (LE) = string of LCs connected by AND 
The design below encodes LC1 AND LC2 AND LC3 

<center> [[Image:logical expression2.GIF]] </center>

'''Subroutine'''

1. Individual bacteral cells use Hin/hix system to randomly choose of of the 64 possible combinations of 6 inputs. Suppressors a and g represent 1 and 0 for the first input; suppressors b and h are 1 and 0 for the second input; etc. up to the sixth input with suppressors f and l (lower case L). The triangles are hix sites for Hin recombination. Whichever of the two suppressor tRNAs in an input pair is facing forward determines whether the value of that input is 1 or 0.

<center> [[Image:suppressor inputs.GIF]] </center>

2. Each bacterial cell carries out the following subroutine on each LC: IF LC=TRUE THEN "check the next LC" ELSEIF LC=FALSE "go get a new set of inputs with step 1" In order to do this, each of the activators below must turn on a repressor that turns off Hin production. Then if one of the activators is not made, Hin will be made, and new inputs will be established.

<center> [[Image:LE circuit.GIF]] </center>

3. If/when a bacterial cell finds a set of inputs that satisfies the entire LE (ie. a solution to the 3-SAT problem), it will glow green.

== Things we need to learn about ==

'''Note: We would need 12 different frameshift suppressor tRNAs to encode the Sakamoto 3-SAT problem'''

'''Discovery of frameshift tRNAs. How many are known?''' 

[http://gcat.davidson.edu/GcatWiki/images/5/59/MaglierySchultz2001JMB.pdf Magliery, Anderson, Schultz]

Library approach used to discover efficient suppressors of four-base codons AGGA, UAGA, CCCU, and CUAG using mutated versions of serine tRNA.

[http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf Anderson, Maglieri, Schultz]

Signals for translational bypassing (slipping and hopping): mRNA secondary structure, "hungry" (underused) codons, upstream Shine-Dalgarno-like (RBS) sequences 
Library approach extended in order to discover frameshift suppressor tRNAs with anticodons of size two to six bases. Two-base and six-base suppressors were not found, but the following five-base suppressors were found:

<center> [[Image:Table2_Anderson2002.GIF]] </center>

[http://gcat.davidson.edu/GcatWiki/images/a/ac/Dunham_2009_tRNA_structure.pdf Dunham et al.]

[http://gcat.davidson.edu/GcatWiki/images/c/c3/Hohsaka_2009_in_vitro_tln.pdf Hohsaka et al.]

'''Processing of tRNA precursors in E. coli''' 

[http://www.nature.com/embor/journal/v2/n1/full/embor501.html Mörl and Marchfelder] describe processing 

<center> [[Image:the_final_cut.GIF]] </center>

'''How many different frameshift suppressor tRNAs can be in one cell without causing toxicity?'''

Can we have twelve of them? If not, what is the maximum? Is there an impact of codon usage on this?

Can we have both 4- and 5-base suppressor tRNAs in one cell?

Are there particular combinations of suppressors that are more tolerated than others?

 
'''How many frameshift mutations can be suppressed in a single gene?'''

How close together can the frameshift mutations of a given LC be?

Can we mix 4- and 5-base frameshift mutations in a given LC?

Can we solve a 3-SAT problem with supressor logic?

2009-05-12T15:12:42Z

Brs3298: /* What is the 3-SAT problem? */

== What is the 3-SAT problem? ==

Advantages/Disadvantages to using the 3 SAT problem

== How did Sakamoto et al. use a DNA computer to solve a 3-SAT problem? ==

[http://gcat.davidson.edu/GcatWiki/images/3/39/Sakamoto_DNA_computer_3SAT.pdf Sakamoto et al.]

<center> [[Image:hairpin2.GIF]] </center>
Sakamoto used hairpin formations in single stranded DNA (ssDNA) as a molecular computer. Hairpin structures are formed when complimentary bases on the same strand attach to each other forming a loop. As shown in Picture B above, C and -C are compliments of each other, and bind together. Picture A is what a normal ssDNA should look like. Sakamoto used this self assembly of secondary structures on a satisfiability (SAT) problem. The problem had six inputs and ten clauses. An example of a clause would be (a or b or -c), and could include any combinations of inputs from a to f including -a to -f. If the problem is satisfied, the ssDNA stays in it's normal form. If the problem is not satisfied, the ssDNA forms a hairpin. Later in the paper it explains that the hairpin forming molecules can be removed from the others with certain techniques. 

The 10 clause 3-SAT problem solved in the paper is:

(a or b or –c) 
(a or c or d) 
(a or –c or –d) 
(-a or –c or d) 
(a or –c or e) 
(a or d or –f) 
(-a or c or d) 
(a or c or –d) 
(-a or –c or –d) 
(-a or c or –d) 

note: this problem uses inputs of a, b, c, d, e, -a, -c, -d, -f (f, -b, -e are not used)

== What is suppressor logic? ==

'''Suppressor Logic uses suppressor tRNAs as inputs to avoid frameshift mutations in the production of an output amino acid sequence'''

A frameshift is a genetic mutation caused by the addition or deletion of nucelotides to a given sequence which codes for a protein. Since codons are read in a series of three, the addition or deletion of nucleotides will disrupt the reading frame. This disruption will most likely cause the production of a nonfunctional protein.

<center> [[Image:suppressor4.GIF]] </center>

A frameshift occurs and, in this case, a guanine is added to the sequence. If nothing is done, enzyme A will not be made, meaning the clause will not be satisfied.

The suppressor tRNA allows the 4 letter sequence to be read as a single codon, therefore, keeping the protein on track. 

If more than one frameshift mutation is introduced into a coding sequence, then logical operators can be encoded. Suppressor a binds to CCCG, supressor b binds to CUGC, and suppressor c binds to ACCG below: 

# AUG CCCG CUG ... rest of Gene 
# AUG CCCG CUGC CUG ... rest of Gene 
# AUG CCCG gg CUGC AGG ... rest of Gene 
# AUG CCCG gg CUGC gg ACCG AGG ... rest of Gene 
 
{| align="center" border="1" cellpadding="8" cellspacing="0" style="background:lightcyan; font-weight:bold; text-align:center; width:45%"
|+
|- style="background:paleturquoise"
! style="width:15%" | Construct
! style="width:15%" | Gene
! style="width:15%" | Logical operation
|-
| 1 || Phenotype || a
|-
| 2 || Phenotype || a AND b
|-
| 3 || Phenotype || a OR b
|-
| 4 || Phenotype || a OR b OR c
|-
| 1 || Repressor|| NOT a
|-
| 2 || Repressor|| a NAND b
|-
| 3 || Repressor|| a NOR b
|-
| 4 || Repressor|| NOT (a OR b OR c)
|}

'''Logical clauses can be connected by AND operators if the proteins produced are part of a biochemical pathway.''' In this case, a AND b AND c: 

<center> [[Image:suppressor2.GIF]] </center>

== How could suppressor logic be used to solve the Sakamoto 3-SAT problem? ==

'''Definitions'''

Inputs = framshift suppressor tRNAs

Input value = supp a is 1, supp g is 0; supp b is 1, supp h is 0, etc. up to tth 6th pair of f and l

Logical clause (LC) = three inputs connected by OR, eg. (a OR b OR e)

LC using 4-base frameshift mutations: cag CCCG aa GGGC tt GTTG cag (may also have any multiple of 3 bases between the mutations) 

LC using 5-base frameshift mutations: cag CCCGC a GGGCG t GTTGC cag (may also have any multiple of 3 bases between the mutations) 

This makes an XOR logic gate as only one suppressor can be used at one time to maintain the reading frame.

Logical expression (LE) = string of LCs connected by AND 
The design below encodes LC1 AND LC2 AND LC3 

<center> [[Image:logical expression2.GIF]] </center>

'''Subroutine'''

1. Individual bacteral cells use Hin/hix system to randomly choose of of the 64 possible combinations of 6 inputs. Suppressors a and g represent 1 and 0 for the first input; suppressors b and h are 1 and 0 for the second input; etc. up to the sixth input with suppressors f and l (lower case L). The triangles are hix sites for Hin recombination. Whichever of the two suppressor tRNAs in an input pair is facing forward determines whether the value of that input is 1 or 0.

<center> [[Image:suppressor inputs.GIF]] </center>

2. Each bacterial cell carries out the following subroutine on each LC: IF LC=TRUE THEN "check the next LC" ELSEIF LC=FALSE "go get a new set of inputs with step 1" In order to do this, each of the activators below must turn on a repressor that turns off Hin production. Then if one of the activators is not made, Hin will be made, and new inputs will be established.

<center> [[Image:LE circuit.GIF]] </center>

3. If/when a bacterial cell finds a set of inputs that satisfies the entire LE (ie. a solution to the 3-SAT problem), it will glow green.

== Things we need to learn about ==

'''Note: We would need 12 different frameshift suppressor tRNAs to encode the Sakamoto 3-SAT problem'''

'''Discovery of frameshift tRNAs. How many are known?''' 

[http://gcat.davidson.edu/GcatWiki/images/5/59/MaglierySchultz2001JMB.pdf Magliery, Anderson, Schultz]

Library approach used to discover efficient suppressors of four-base codons AGGA, UAGA, CCCU, and CUAG using mutated versions of serine tRNA.

[http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf Anderson, Maglieri, Schultz]

Signals for translational bypassing (slipping and hopping): mRNA secondary structure, "hungry" (underused) codons, upstream Shine-Dalgarno-like (RBS) sequences 
Library approach extended in order to discover frameshift suppressor tRNAs with anticodons of size two to six bases. Two-base and six-base suppressors were not found, but the following five-base suppressors were found:

<center> [[Image:Table2_Anderson2002.GIF]] </center>

[http://gcat.davidson.edu/GcatWiki/images/a/ac/Dunham_2009_tRNA_structure.pdf Dunham et al.]

[http://gcat.davidson.edu/GcatWiki/images/c/c3/Hohsaka_2009_in_vitro_tln.pdf Hohsaka et al.]

'''Processing of tRNA precursors in E. coli''' 

[http://www.nature.com/embor/journal/v2/n1/full/embor501.html Mörl and Marchfelder] describe processing 

<center> [[Image:the_final_cut.GIF]] </center>

'''How many different frameshift suppressor tRNAs can be in one cell without causing toxicity?'''

Can we have twelve of them? If not, what is the maximum? Is there an impact of codon usage on this?

Can we have both 4- and 5-base suppressor tRNAs in one cell?

Are there particular combinations of suppressors that are more tolerated than others?

 
'''How many frameshift mutations can be suppressed in a single gene?'''

How close together can the frameshift mutations of a given LC be?

Can we mix 4- and 5-base frameshift mutations in a given LC?

Can we solve a 3-SAT problem with supressor logic?

2009-05-12T15:12:09Z

Brs3298: /* Advantages/Disadvantages to the 3 SAT problem */

== What is the 3-SAT problem? ==

== How did Sakamoto et al. use a DNA computer to solve a 3-SAT problem? ==

[http://gcat.davidson.edu/GcatWiki/images/3/39/Sakamoto_DNA_computer_3SAT.pdf Sakamoto et al.]

<center> [[Image:hairpin2.GIF]] </center>
Sakamoto used hairpin formations in single stranded DNA (ssDNA) as a molecular computer. Hairpin structures are formed when complimentary bases on the same strand attach to each other forming a loop. As shown in Picture B above, C and -C are compliments of each other, and bind together. Picture A is what a normal ssDNA should look like. Sakamoto used this self assembly of secondary structures on a satisfiability (SAT) problem. The problem had six inputs and ten clauses. An example of a clause would be (a or b or -c), and could include any combinations of inputs from a to f including -a to -f. If the problem is satisfied, the ssDNA stays in it's normal form. If the problem is not satisfied, the ssDNA forms a hairpin. Later in the paper it explains that the hairpin forming molecules can be removed from the others with certain techniques. 

The 10 clause 3-SAT problem solved in the paper is:

(a or b or –c) 
(a or c or d) 
(a or –c or –d) 
(-a or –c or d) 
(a or –c or e) 
(a or d or –f) 
(-a or c or d) 
(a or c or –d) 
(-a or –c or –d) 
(-a or c or –d) 

note: this problem uses inputs of a, b, c, d, e, -a, -c, -d, -f (f, -b, -e are not used)

== What is suppressor logic? ==

'''Suppressor Logic uses suppressor tRNAs as inputs to avoid frameshift mutations in the production of an output amino acid sequence'''

A frameshift is a genetic mutation caused by the addition or deletion of nucelotides to a given sequence which codes for a protein. Since codons are read in a series of three, the addition or deletion of nucleotides will disrupt the reading frame. This disruption will most likely cause the production of a nonfunctional protein.

<center> [[Image:suppressor4.GIF]] </center>

A frameshift occurs and, in this case, a guanine is added to the sequence. If nothing is done, enzyme A will not be made, meaning the clause will not be satisfied.

The suppressor tRNA allows the 4 letter sequence to be read as a single codon, therefore, keeping the protein on track. 

If more than one frameshift mutation is introduced into a coding sequence, then logical operators can be encoded. Suppressor a binds to CCCG, supressor b binds to CUGC, and suppressor c binds to ACCG below: 

# AUG CCCG CUG ... rest of Gene 
# AUG CCCG CUGC CUG ... rest of Gene 
# AUG CCCG gg CUGC AGG ... rest of Gene 
# AUG CCCG gg CUGC gg ACCG AGG ... rest of Gene 
 
{| align="center" border="1" cellpadding="8" cellspacing="0" style="background:lightcyan; font-weight:bold; text-align:center; width:45%"
|+
|- style="background:paleturquoise"
! style="width:15%" | Construct
! style="width:15%" | Gene
! style="width:15%" | Logical operation
|-
| 1 || Phenotype || a
|-
| 2 || Phenotype || a AND b
|-
| 3 || Phenotype || a OR b
|-
| 4 || Phenotype || a OR b OR c
|-
| 1 || Repressor|| NOT a
|-
| 2 || Repressor|| a NAND b
|-
| 3 || Repressor|| a NOR b
|-
| 4 || Repressor|| NOT (a OR b OR c)
|}

'''Logical clauses can be connected by AND operators if the proteins produced are part of a biochemical pathway.''' In this case, a AND b AND c: 

<center> [[Image:suppressor2.GIF]] </center>

== How could suppressor logic be used to solve the Sakamoto 3-SAT problem? ==

'''Definitions'''

Inputs = framshift suppressor tRNAs

Input value = supp a is 1, supp g is 0; supp b is 1, supp h is 0, etc. up to tth 6th pair of f and l

Logical clause (LC) = three inputs connected by OR, eg. (a OR b OR e)

LC using 4-base frameshift mutations: cag CCCG aa GGGC tt GTTG cag (may also have any multiple of 3 bases between the mutations) 

LC using 5-base frameshift mutations: cag CCCGC a GGGCG t GTTGC cag (may also have any multiple of 3 bases between the mutations) 

This makes an XOR logic gate as only one suppressor can be used at one time to maintain the reading frame.

Logical expression (LE) = string of LCs connected by AND 
The design below encodes LC1 AND LC2 AND LC3 

<center> [[Image:logical expression2.GIF]] </center>

'''Subroutine'''

1. Individual bacteral cells use Hin/hix system to randomly choose of of the 64 possible combinations of 6 inputs. Suppressors a and g represent 1 and 0 for the first input; suppressors b and h are 1 and 0 for the second input; etc. up to the sixth input with suppressors f and l (lower case L). The triangles are hix sites for Hin recombination. Whichever of the two suppressor tRNAs in an input pair is facing forward determines whether the value of that input is 1 or 0.

<center> [[Image:suppressor inputs.GIF]] </center>

2. Each bacterial cell carries out the following subroutine on each LC: IF LC=TRUE THEN "check the next LC" ELSEIF LC=FALSE "go get a new set of inputs with step 1" In order to do this, each of the activators below must turn on a repressor that turns off Hin production. Then if one of the activators is not made, Hin will be made, and new inputs will be established.

<center> [[Image:LE circuit.GIF]] </center>

3. If/when a bacterial cell finds a set of inputs that satisfies the entire LE (ie. a solution to the 3-SAT problem), it will glow green.

== Things we need to learn about ==

'''Note: We would need 12 different frameshift suppressor tRNAs to encode the Sakamoto 3-SAT problem'''

'''Discovery of frameshift tRNAs. How many are known?''' 

[http://gcat.davidson.edu/GcatWiki/images/5/59/MaglierySchultz2001JMB.pdf Magliery, Anderson, Schultz]

Library approach used to discover efficient suppressors of four-base codons AGGA, UAGA, CCCU, and CUAG using mutated versions of serine tRNA.

[http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf Anderson, Maglieri, Schultz]

Signals for translational bypassing (slipping and hopping): mRNA secondary structure, "hungry" (underused) codons, upstream Shine-Dalgarno-like (RBS) sequences 
Library approach extended in order to discover frameshift suppressor tRNAs with anticodons of size two to six bases. Two-base and six-base suppressors were not found, but the following five-base suppressors were found:

<center> [[Image:Table2_Anderson2002.GIF]] </center>

[http://gcat.davidson.edu/GcatWiki/images/a/ac/Dunham_2009_tRNA_structure.pdf Dunham et al.]

[http://gcat.davidson.edu/GcatWiki/images/c/c3/Hohsaka_2009_in_vitro_tln.pdf Hohsaka et al.]

'''Processing of tRNA precursors in E. coli''' 

[http://www.nature.com/embor/journal/v2/n1/full/embor501.html Mörl and Marchfelder] describe processing 

<center> [[Image:the_final_cut.GIF]] </center>

'''How many different frameshift suppressor tRNAs can be in one cell without causing toxicity?'''

Can we have twelve of them? If not, what is the maximum? Is there an impact of codon usage on this?

Can we have both 4- and 5-base suppressor tRNAs in one cell?

Are there particular combinations of suppressors that are more tolerated than others?

 
'''How many frameshift mutations can be suppressed in a single gene?'''

How close together can the frameshift mutations of a given LC be?

Can we mix 4- and 5-base frameshift mutations in a given LC?

Can we solve a 3-SAT problem with supressor logic?

2009-05-12T15:11:48Z

Brs3298: /* What is the 3-SAT problem? */

== What is the 3-SAT problem? ==

= Advantages/Disadvantages to the 3 SAT problem =

== How did Sakamoto et al. use a DNA computer to solve a 3-SAT problem? ==

[http://gcat.davidson.edu/GcatWiki/images/3/39/Sakamoto_DNA_computer_3SAT.pdf Sakamoto et al.]

<center> [[Image:hairpin2.GIF]] </center>
Sakamoto used hairpin formations in single stranded DNA (ssDNA) as a molecular computer. Hairpin structures are formed when complimentary bases on the same strand attach to each other forming a loop. As shown in Picture B above, C and -C are compliments of each other, and bind together. Picture A is what a normal ssDNA should look like. Sakamoto used this self assembly of secondary structures on a satisfiability (SAT) problem. The problem had six inputs and ten clauses. An example of a clause would be (a or b or -c), and could include any combinations of inputs from a to f including -a to -f. If the problem is satisfied, the ssDNA stays in it's normal form. If the problem is not satisfied, the ssDNA forms a hairpin. Later in the paper it explains that the hairpin forming molecules can be removed from the others with certain techniques. 

The 10 clause 3-SAT problem solved in the paper is:

(a or b or –c) 
(a or c or d) 
(a or –c or –d) 
(-a or –c or d) 
(a or –c or e) 
(a or d or –f) 
(-a or c or d) 
(a or c or –d) 
(-a or –c or –d) 
(-a or c or –d) 

note: this problem uses inputs of a, b, c, d, e, -a, -c, -d, -f (f, -b, -e are not used)

== What is suppressor logic? ==

'''Suppressor Logic uses suppressor tRNAs as inputs to avoid frameshift mutations in the production of an output amino acid sequence'''

A frameshift is a genetic mutation caused by the addition or deletion of nucelotides to a given sequence which codes for a protein. Since codons are read in a series of three, the addition or deletion of nucleotides will disrupt the reading frame. This disruption will most likely cause the production of a nonfunctional protein.

<center> [[Image:suppressor4.GIF]] </center>

A frameshift occurs and, in this case, a guanine is added to the sequence. If nothing is done, enzyme A will not be made, meaning the clause will not be satisfied.

The suppressor tRNA allows the 4 letter sequence to be read as a single codon, therefore, keeping the protein on track. 

If more than one frameshift mutation is introduced into a coding sequence, then logical operators can be encoded. Suppressor a binds to CCCG, supressor b binds to CUGC, and suppressor c binds to ACCG below: 

# AUG CCCG CUG ... rest of Gene 
# AUG CCCG CUGC CUG ... rest of Gene 
# AUG CCCG gg CUGC AGG ... rest of Gene 
# AUG CCCG gg CUGC gg ACCG AGG ... rest of Gene 
 
{| align="center" border="1" cellpadding="8" cellspacing="0" style="background:lightcyan; font-weight:bold; text-align:center; width:45%"
|+
|- style="background:paleturquoise"
! style="width:15%" | Construct
! style="width:15%" | Gene
! style="width:15%" | Logical operation
|-
| 1 || Phenotype || a
|-
| 2 || Phenotype || a AND b
|-
| 3 || Phenotype || a OR b
|-
| 4 || Phenotype || a OR b OR c
|-
| 1 || Repressor|| NOT a
|-
| 2 || Repressor|| a NAND b
|-
| 3 || Repressor|| a NOR b
|-
| 4 || Repressor|| NOT (a OR b OR c)
|}

'''Logical clauses can be connected by AND operators if the proteins produced are part of a biochemical pathway.''' In this case, a AND b AND c: 

<center> [[Image:suppressor2.GIF]] </center>

== How could suppressor logic be used to solve the Sakamoto 3-SAT problem? ==

'''Definitions'''

Inputs = framshift suppressor tRNAs

Input value = supp a is 1, supp g is 0; supp b is 1, supp h is 0, etc. up to tth 6th pair of f and l

Logical clause (LC) = three inputs connected by OR, eg. (a OR b OR e)

LC using 4-base frameshift mutations: cag CCCG aa GGGC tt GTTG cag (may also have any multiple of 3 bases between the mutations) 

LC using 5-base frameshift mutations: cag CCCGC a GGGCG t GTTGC cag (may also have any multiple of 3 bases between the mutations) 

This makes an XOR logic gate as only one suppressor can be used at one time to maintain the reading frame.

Logical expression (LE) = string of LCs connected by AND 
The design below encodes LC1 AND LC2 AND LC3 

<center> [[Image:logical expression2.GIF]] </center>

'''Subroutine'''

1. Individual bacteral cells use Hin/hix system to randomly choose of of the 64 possible combinations of 6 inputs. Suppressors a and g represent 1 and 0 for the first input; suppressors b and h are 1 and 0 for the second input; etc. up to the sixth input with suppressors f and l (lower case L). The triangles are hix sites for Hin recombination. Whichever of the two suppressor tRNAs in an input pair is facing forward determines whether the value of that input is 1 or 0.

<center> [[Image:suppressor inputs.GIF]] </center>

2. Each bacterial cell carries out the following subroutine on each LC: IF LC=TRUE THEN "check the next LC" ELSEIF LC=FALSE "go get a new set of inputs with step 1" In order to do this, each of the activators below must turn on a repressor that turns off Hin production. Then if one of the activators is not made, Hin will be made, and new inputs will be established.

<center> [[Image:LE circuit.GIF]] </center>

3. If/when a bacterial cell finds a set of inputs that satisfies the entire LE (ie. a solution to the 3-SAT problem), it will glow green.

== Things we need to learn about ==

'''Note: We would need 12 different frameshift suppressor tRNAs to encode the Sakamoto 3-SAT problem'''

'''Discovery of frameshift tRNAs. How many are known?''' 

[http://gcat.davidson.edu/GcatWiki/images/5/59/MaglierySchultz2001JMB.pdf Magliery, Anderson, Schultz]

Library approach used to discover efficient suppressors of four-base codons AGGA, UAGA, CCCU, and CUAG using mutated versions of serine tRNA.

[http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf Anderson, Maglieri, Schultz]

Signals for translational bypassing (slipping and hopping): mRNA secondary structure, "hungry" (underused) codons, upstream Shine-Dalgarno-like (RBS) sequences 
Library approach extended in order to discover frameshift suppressor tRNAs with anticodons of size two to six bases. Two-base and six-base suppressors were not found, but the following five-base suppressors were found:

<center> [[Image:Table2_Anderson2002.GIF]] </center>

[http://gcat.davidson.edu/GcatWiki/images/a/ac/Dunham_2009_tRNA_structure.pdf Dunham et al.]

[http://gcat.davidson.edu/GcatWiki/images/c/c3/Hohsaka_2009_in_vitro_tln.pdf Hohsaka et al.]

'''Processing of tRNA precursors in E. coli''' 

[http://www.nature.com/embor/journal/v2/n1/full/embor501.html Mörl and Marchfelder] describe processing 

<center> [[Image:the_final_cut.GIF]] </center>

'''How many different frameshift suppressor tRNAs can be in one cell without causing toxicity?'''

Can we have twelve of them? If not, what is the maximum? Is there an impact of codon usage on this?

Can we have both 4- and 5-base suppressor tRNAs in one cell?

Are there particular combinations of suppressors that are more tolerated than others?

 
'''How many frameshift mutations can be suppressed in a single gene?'''

How close together can the frameshift mutations of a given LC be?

Can we mix 4- and 5-base frameshift mutations in a given LC?

File:RFP.GIF

2009-05-11T16:04:03Z

Brs3298:

What other cool reporters are there? (Discrete On/Off or Continuous) Bryce Szczepanik

2009-05-11T16:03:25Z

Brs3298: /* Color */

=== What is a reporter? ===

A reporter is a gene that can be attached to another gene of interest in DNA. Certain genes are chosen as reporters because the characteristics that they confer on the organism that is expressing them are easy to measure and see. Reporter genes are usually used as a marker to determine if a given gene has been expressed in an organism.

=== Examples of reporters ===

== Color ==

With all of the different color outputs available (a few are shown below) each different gene could be coded for by a different color.

Part: BBa_I13521 is RFP or red fluorescent protein. When visualized under UV light, a luminescent red color can be observed in organisms exhibiting the gene.

Part: BBa_E0240 is GFP or green fluorescent protein.

Part : BBa_I13600 is CFP or cyan fluorescent protein. In the absence of the tetR protein, CFP expression is constitutive. tetR represses CFP production; this repression can be relieved by the addition of tetracycline or one of its analogs (ie. aTc).

Part: BBa_E0430 is YFP or yellow fluorescent protein.

<center> [[Image:RFP.GIF]] </center>

== Antibiotic resistance ==

The bacterial colonies can be grown on an agar plate that contains an antibiotic such as ampicillin or tetracycline. A reporter gene can be used to code for resistance to an antibiotic. This is a neat reporter that is easy to visualize. If the bacteria are growing on the antibiotic plate, they are resistant and, therefore, contain the reporter gene that codes for that resistance.

== Lactose and Tryptophan ==

== Others ==

What other cool reporters are there? (Discrete On/Off or Continuous) Bryce Szczepanik

2009-05-11T15:58:34Z

Brs3298: /* Color */

=== What is a reporter? ===

A reporter is a gene that can be attached to another gene of interest in DNA. Certain genes are chosen as reporters because the characteristics that they confer on the organism that is expressing them are easy to measure and see. Reporter genes are usually used as a marker to determine if a given gene has been expressed in an organism.

=== Examples of reporters ===

== Color ==

With all of the different color outputs available (a few are shown below) each different gene could be coded for by a different color.

Part: BBa_I13521 is RFP or red fluorescent protein. When visualized under UV light, a luminescent red color can be observed in organisms exhibiting the gene.

Part: BBa_E0240 is GFP or green fluorescent protein.

Part : BBa_I13600 is CFP or cyan fluorescent protein. In the absence of the tetR protein, CFP expression is constitutive. tetR represses CFP production; this repression can be relieved by the addition of tetracycline or one of its analogs (ie. aTc).

Part: BBa_E0430 is YFP or yellow fluorescent protein.

== Antibiotic resistance ==

The bacterial colonies can be grown on an agar plate that contains an antibiotic such as ampicillin or tetracycline. A reporter gene can be used to code for resistance to an antibiotic. This is a neat reporter that is easy to visualize. If the bacteria are growing on the antibiotic plate, they are resistant and, therefore, contain the reporter gene that codes for that resistance.

== Lactose and Tryptophan ==

== Others ==

What other cool reporters are there? (Discrete On/Off or Continuous) Bryce Szczepanik

2009-05-11T15:57:09Z

Brs3298: /* Some reporters */

=== What is a reporter? ===

A reporter is a gene that can be attached to another gene of interest in DNA. Certain genes are chosen as reporters because the characteristics that they confer on the organism that is expressing them are easy to measure and see. Reporter genes are usually used as a marker to determine if a given gene has been expressed in an organism.

=== Examples of reporters ===

== Color ==

With all of the different color outputs available (a few are shown below) each different gene could be coded for by a different color.
Part: BBa_I13521 is RFP or red fluorescent protein. When visualized under UV light, a luminescent red color can be observed in organisms exhibiting the gene.
Part: BBa_E0240 is GFP or green fluorescent protein.
Part : BBa_I13600 is CFP or cyan fluorescent protein. In the absence of the tetR protein, CFP expression is constitutive. tetR represses CFP production; this repression can be relieved by the addition of tetracycline or one of its analogs (ie. aTc).
Part: BBa_E0430 is YFP or yellow fluorescent protein.
Antibiotic resistance
The bacterial colonies can be grown on an agar plate that contains an antibiotic such as ampicillin or tetracycline. A reporter gene can be used to code for resistance to an antibiotic. This is a neat reporter that is easy to visualize. If the bacteria are growing on the antibiotic plate, they are resistant and, therefore, contain the reporter gene that codes for that resistance.

== Lactose and Tryptophan ==

== Others ==

What other cool reporters are there? (Discrete On/Off or Continuous) Bryce Szczepanik

2009-05-11T15:56:11Z

Brs3298:

=== What is a reporter? ===

A reporter is a gene that can be attached to another gene of interest in DNA. Certain genes are chosen as reporters because the characteristics that they confer on the organism that is expressing them are easy to measure and see. Reporter genes are usually used as a marker to determine if a given gene has been expressed in an organism.

=== Some reporters ===

== Color ==

With all of the different color outputs available (a few are shown below) each different gene could be coded for by a different color.
Part: BBa_I13521 is RFP or red fluorescent protein. When visualized under UV light, a luminescent red color can be observed in organisms exhibiting the gene.
Part: BBa_E0240 is GFP or green fluorescent protein.
Part : BBa_I13600 is CFP or cyan fluorescent protein. In the absence of the tetR protein, CFP expression is constitutive. tetR represses CFP production; this repression can be relieved by the addition of tetracycline or one of its analogs (ie. aTc).
Part: BBa_E0430 is YFP or yellow fluorescent protein.
Antibiotic resistance
The bacterial colonies can be grown on an agar plate that contains an antibiotic such as ampicillin or tetracycline. A reporter gene can be used to code for resistance to an antibiotic. This is a neat reporter that is easy to visualize. If the bacteria are growing on the antibiotic plate, they are resistant and, therefore, contain the reporter gene that codes for that resistance.

== Lactose and Tryptophan ==

== Others ==

Can we solve a 3-SAT problem with supressor logic?

2009-04-07T20:54:12Z

Brs3298: /* What is suppressor logic? */

File:TRNAandFSM.GIF

2009-04-07T20:41:02Z

Brs3298:

Can we solve a 3-SAT problem with supressor logic?

2009-04-07T20:40:52Z

Brs3298: /* What is suppressor logic? */

File:Suppressor4.GIF

2009-04-07T20:32:03Z

Brs3298:

Can we solve a 3-SAT problem with supressor logic?

2009-04-07T20:31:50Z

Brs3298: /* What is suppressor logic? */

File:Suppressor3.GIF

2009-04-07T20:29:28Z

Brs3298:

Can we solve a 3-SAT problem with supressor logic?

2009-04-07T20:29:12Z

Brs3298: /* What is suppressor logic? */

File:Suppressor2.GIF

2009-04-07T20:11:15Z

Brs3298:

Can we solve a 3-SAT problem with supressor logic?

2009-04-07T20:11:05Z

Brs3298: /* What is suppressor logic? */

File:Suppressor1.GIF

2009-04-07T20:05:16Z

Brs3298:

Can we solve a 3-SAT problem with supressor logic?

2009-04-07T20:04:54Z

Brs3298: /* What is suppressor logic? */