Difference between revisions of "Present Hybrid Promoter Designs cartoon fashion (3 major different types)"

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[[Oligos_to_Build]]: Sequences we will need to make this XOR gate.
 
[[Oligos_to_Build]]: Sequences we will need to make this XOR gate.
  
''LIST OF Km Values''<br>
 
*Km of '''3OC12 for LasR''' is 1 nM ([http://www.bio.davidson.edu/courses/synthetic/papers/LuxR.pdf Egland and Greenberg, 2000]) <br>
 
*Km of '''3OC6 for LuxR''' is 100 nM ([http://www.bio.davidson.edu/courses/synthetic/papers/Urbanowski_04.pdf Urbanowski et al., 2004])<br>
 
*Km of '''LasR* for operator/promoter''' is 11 pM ([http://www.bio.davidson.edu/courses/synthetic/papers/Schuster_04.pdf Schuster et al, 2004])<br>
 
*Km of '''LuxR* for operator/promoter''' is 10 nM ([http://www.bio.davidson.edu/courses/synthetic/papers/Kapland_Greenberg_87.pdf Kaplan and Greenberg, 1987])<br>
 
*Km of '''cI dimer for OR1 and OR2''' is 10 pM ([http://www.bio.davidson.edu/courses/synthetic/papers/King_93.pdf King et al., 1993])<br>
 
*Km of '''Mnt tetramer for binding to half operator/promoter''' is 50 nM  and '''binding whole operator''' is 1 nM ([http://www.bio.davidson.edu/courses/synthetic/papers/Berggrun_01.pdf Berggrun and Sauer, 2001])<br>
 
*Km of '''Lsr for its binding site''' is X nM (no data available) <br>
 
*Km of '''AI-2 for LsrR''' is X nM (no data available) <br>
 
*Km of '''IPTG for LacI''' is 1.3 µM ([http://www.bio.davidson.edu/courses/synthetic/papers/Gibert_Muller_hill_66.pdf Gilbert and Muller-Hill, 1966]) <br>
 
*Km of '''LacI for its binding site''' is 10 pM ([http://www.bio.davidson.edu/courses/synthetic/papers/Gilbert_Muller_Hill_67.pdf Gilbert and Muller-Hill, 1967]) <br>
 
*Km of '''LacI-I12 for its binding site''' is 0.13 pM (calculated from [http://www.bio.davidson.edu/courses/synthetic/papers/pLac_1980.pdf Schmitz and Galas, 1980]; and [http://www.bio.davidson.edu/courses/synthetic/papers/Scmitz_etal_78.pdf Schmitz et al., 1978])<br>
 
*Km of '''LacI-X86 for its binding site''' is 0.13 pM (calculated from [http://www.bio.davidson.edu/courses/synthetic/papers/pLac_1980.pdf Schmitz and Galas, 1980]; and [http://www.bio.davidson.edu/courses/synthetic/papers/Scmitz_etal_78.pdf Schmitz et al., 1978])<br>
 
*Km of '''LacI-I12_X86 for its binding site''' is 0.001 pM (calculated from [http://www.bio.davidson.edu/courses/synthetic/papers/pLac_1980.pdf Schmitz and Galas, 1980]; and [http://www.bio.davidson.edu/courses/synthetic/papers/Scmitz_etal_78.pdf Schmitz et al., 1978])<br>
 
*'''Half life of molecules in ''E. coli'':''' 2 minutes for mRNA; 1 molecule is 1 nM concentration ([http://www.bio.davidson.edu/courses/synthetic/papers/lsr_thesis_07.pdf Fang Ph.D. Thesis, 2007]); translation rate 15 amino acids per second and transcription is 40 nt per second (from ''Genes VII'' by Lewin).
 
  
 
== Missouri Western XOR Biological Design 1==
 
== Missouri Western XOR Biological Design 1==
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These two XOR circuits are designed to complement each other. Each recieves a cell-to-cell signal (AI-1 or AI-2) and a chemical signal (IPTG or AHL) and processes it into a cell-to-cell signal.  Colonies that output AI-1 would alternate with colonies that produce AI-2.  The input message to be hashed could be encoded by the presence or absence of the chemical signals, which would also alternate.   
 
These two XOR circuits are designed to complement each other. Each recieves a cell-to-cell signal (AI-1 or AI-2) and a chemical signal (IPTG or AHL) and processes it into a cell-to-cell signal.  Colonies that output AI-1 would alternate with colonies that produce AI-2.  The input message to be hashed could be encoded by the presence or absence of the chemical signals, which would also alternate.   
  
'''Design Variables'''
 
  
1. strength on RBS for each of the coding sequences (eg. RBS for enzymes could be weak)
 
 
2. order of coding sequences (eg. enzymes could be second for lower expression level)
 
 
3. identity of repressors ('''Davidson is building three different LacI repressors''')
 
 
4. type of degradation tag on proteins
 
 
'''Things to Do'''
 
 
1. make a list of the parts needed and their building status
 
 
2. design, order, and clone 4 hybrid promoters
 
 
3. test the 4 hybrid promoters with GFP or RFP outputs
 
 
 
'''[[DNA Sequences of Relevant Parts]]
 
'''
 
 
'''[[Parts from the registry]]
 
'''
 
 
'''[[Oligo design for XOR Hybrid Promoters]]
 
 
'''[[Parts needed for XOR]]
 
 
'''[[Build XOR Gate]]
 
 
'''[[Test XOR Hybrid Promoters]]
 
 
'''[[Needed From Davidson]]'''
 
  
 
[[Image:XOR DR AI2.PNG]]
 
[[Image:XOR DR AI2.PNG]]

Latest revision as of 14:58, 28 October 2008

Davidson XOR Biological Design

List of auto-inducers and their catalog numbers.

Davidson Approach

Here is an idea Malcolm and Laurie developed.

Everyone please look at this and ask questions and find holes in it now so we don't waste time building something that won't work.
XOR AMC1b.jpg

The idea is to have two mirrored halves of the system. LasR is regulated by PAI-1 {3-oxododecanoyl-HSL (3OC12HSL)} and LuxR is activated by AI-1 {3-oxohexanoyl-homoserine lactone (3OC6HSL)}. There is a potential problem in that the Lux half is more likely to get positive feedback than the Las half. This MAY not be a problem because 0/0 is leaky so we put a weak RBS to minimize leaky protein production. Also, if we add AI-2 and AI-1 is produced by leak, then the entire system shuts down. The repressor site is located between -35 and -10 of the promoter. The activator binding site is upstream of -35. This has been documented by Egland and Greenberg

Oligos_to_Build: Sequences we will need to make this XOR gate.


Missouri Western XOR Biological Design 1

These two XOR circuits are designed to complement each other. Each recieves a cell-to-cell signal (AI-1 or AI-2) and a chemical signal (IPTG or AHL) and processes it into a cell-to-cell signal. Colonies that output AI-1 would alternate with colonies that produce AI-2. The input message to be hashed could be encoded by the presence or absence of the chemical signals, which would also alternate.


XOR DR AI2.PNG

Above - Input of AI-1 or IPTG turns on production of AI-2 by LuxS. Input by both AI-1 and IPTG allows production of the repressors cI and Mnt, which repress both transcription units. LuxR and LacI are constitutively expressed.


XOR DR AI1b.PNG
Above - Input of AI-2 or aTc turns on production of AI-1 by LuxI. Input by both AI-2 and aTc allows production of the repressors cI and Mnt, which repress both transcription units. LsrK, LsrR and TetR are constitutively expressed.