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Team:Aix-Marseille/Project
From 2014.igem.org
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<h2 class="subtitle">References</h2> | <h2 class="subtitle">References</h2> | ||
<p>Pertinent articles related to our project:</p> | <p>Pertinent articles related to our project:</p> | ||
| - | <h3 class=" | + | <h3 class="text-info">Top Papers</h2> |
<ul> | <ul> | ||
<li>Gudipaty SA1, Larsen AS, Rensing C, McEvoy MM. Regulation of Cu(I)/Ag(I) efflux genes in Escherichia coli by the sensor kinase CusS. FEMS Microbiol Lett. 2012 May;330(1):30-7. </li> | <li>Gudipaty SA1, Larsen AS, Rensing C, McEvoy MM. Regulation of Cu(I)/Ag(I) efflux genes in Escherichia coli by the sensor kinase CusS. FEMS Microbiol Lett. 2012 May;330(1):30-7. </li> | ||
| Line 166: | Line 166: | ||
<li>Peters-Wendisch P1, Stolz M, Etterich H, Kennerknecht N, Sahm H, Eggeling L. Metabolic engineering of Corynebacterium glutamicum for L-serine production. Appl Environ Microbiol. 2005 Nov;71(11):7139-44.</li> | <li>Peters-Wendisch P1, Stolz M, Etterich H, Kennerknecht N, Sahm H, Eggeling L. Metabolic engineering of Corynebacterium glutamicum for L-serine production. Appl Environ Microbiol. 2005 Nov;71(11):7139-44.</li> | ||
</ul> | </ul> | ||
| - | <h3 class=" | + | <h3 class="text-info">Related Papers</h2> |
<ul> | <ul> | ||
| - | <li> | + | <li>Wahl A, My L, Dumoulin R, Sturgis JN, Bouveret E.Antagonistic regulation of dgkA and plsB genes of phospholipid synthesis by multiple stress responses in Escherichia coli. Mol Microbiol. 80(5):1260-75, 2011.</li> |
| - | <li> | + | <li>Collins CH. Cell-cell communication special issue. ACS Synth Biol. 2014 Apr 18;3(4):1978.</li> |
| - | <li> | + | <li>Hsiao V, de Los Santos EL, Whitaker WR, Dueber JE, Murray RM. Design and Implementation of a Biomolecular Concentration Tracker. ACS Synth Biol. 2014 May 15.</li> |
| - | <li> | + | <li>Park JH, Oh JE, Lee KH, Kim JY, Lee SY. Rational design of Escherichia coli for L-isoleucine production. ACS Synth Biol. 2012 Nov 16;1(11):532-40. </li> |
| - | <li> | + | <li>Tan MH1, Kozdon JB, Shen X, Shapiro L, McAdams HH. An essential transcription factor, SciP, enhances robustness of Caulobacter cell cycle regulation. Proc Natl Acad Sci U S A. 2010 Nov 2;107(44):18985-90.</li> |
| + | <li>Park H, Saha SK, Inouye M. Two-domain reconstitution of a functional protein histidine kinase. Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6728-32.</li> | ||
| + | <li>Porter SL, Wadhams GH, Armitage JP. Signal processing in complex chemotaxis pathways.Nat Rev Microbiol. 2011 Mar;9(3):153-65.</li> | ||
| + | <li>Olson EJ, Hartsough LA, Landry BP, Shroff R, Tabor JJ. Characterizing bacterial gene circuit dynamics with optically programmed gene expression signals. Nat Methods. 2014 Apr;11(4):449-55.</li> | ||
| + | <li>Skerker JM1, Perchuk BS, Siryaporn A, Lubin EA, Ashenberg O, Goulian M, Laub MT. Rewiring the specificity of two-component signal transduction systems. Cell. 2008 Jun 13;133(6):1043-54.</li> | ||
| + | <li>Skerker JM1, Prasol MS, Perchuk BS, Biondi EG, Laub MT. Two-component signal transduction pathways regulating growth and cell cycle progression in a bacterium: a system-level analysis. PLoS Biol. 2005 Oct;3(10):e334.</li> | ||
| + | <li>Gonzalez D, Collier J. Effects of (p)ppGpp on the progression of the cell cycle of Caulobacter crescentus. J Bacteriol. 2014 Jul;196(14):2514-25.</li> | ||
| + | <li>Thanbichler M. Synchronization of chromosome dynamics and cell division in bacteria. Cold Spring Harb Perspect Biol. 2010 Jan;2(1):a000331.</li> | ||
| + | <li>Wang X, Vallurupalli P, Vu A, Lee K, Sun S, Bai WJ, Wu C, Zhou H, Shea JE, Kay LE, Dahlquist FW. The linker between the dimerization and catalytic domains of the CheA histidine kinase propagates changes in structure and dynamics that are important for enzymatic activity. Biochemistry. 2014 Feb 11;53(5):855-61.</li> | ||
| + | <li>Eggeling L, Sahm H. New ubiquitous translocators: amino acid export by Corynebacterium glutamicum and Escherichia coli. Arch Microbiol. 2003 Sep;180(3):155-60.</li> | ||
| + | <li>Laub MT, Goulian M. Specificity in two-component signal transduction pathways. Annu Rev Genet. 2007;41:121-45.</li> | ||
| + | <li>Alon U, Camarena L, Surette MG, Aguera y Arcas B, Liu Y, Leibler S, Stock JB. Response regulator output in bacterial chemotaxis. EMBO J. 1998 Aug 3;17(15):4238-48.</li> | ||
| + | <li>Chang YC, Armitage JP, Papachristodoulou A, Wadhams GH. A single phosphatase can convert a robust step response into a graded, tunable or adaptive response. Microbiology. 2013 Jul;159(Pt 7):1276-85.</li> | ||
| + | <li>Ganesh I, Ravikumar S, Lee SH, Park SJ, Hong SH. Engineered fumarate sensing Escherichia coli based on novel chimeric two-component system. J Biotechnol. 2013 Dec;168(4):560-6.</li> | ||
| + | <li>Jonas K. To divide or not to divide: control of the bacterial cell cycle by environmental cues. Curr Opin Microbiol. 2014 Apr;18:54-60.</li> | ||
</ul> | </ul> | ||
</div> | </div> | ||
Revision as of 15:55, 11 October 2014
S.E.colization : Green light, GO!
Overall project summary
Our project centers around synchronizing a culture of E.coli cells, so that they all divide synchronously and change color from green to red and back as they go through the cell cycle. More specifically the culture stops dividing full of red cells and then at regular intervals the cells turn green and divide before returning to the red quiescent state.
This project relies on developing several new and original modules and components which we believe will be generally useful to other teams in future projects. The first component is an inducible Serine production system based on a mutant SerA protein that is insensitive to retro-inhibition by the pathway product (serine) allowing the development of bacteria secreting serine. The second component involves re-engineering the chemotaxis system to drive changes in gene expression rather than changes in flagellar rotation direction. This component is particularly innovative and potentially useful as it can form the basis of several different synthetic signaling system allowing regulation of gene expression by a wide range of different signaling molecules. The third component, or set of components, is a series of switches that change the protein expressed in response to a signal, that is rather than a simple induction of expression as observed with most inducible systems, one protein will be expressed while a second is repressed. Again it is hoped that the bricks that make this system will be applicable in multiple new projects. Each of these components is designed so that if they are introduced together they will produce an oscillator that regularly drives the switch modules between their two states. This oscillator will be coupled to color changes, the initiation of the cell cycle, and serine production (to make the feedback loop). A particularity of the oscillator is that by passing through a secreted substrate and sensor system the oscillations should be culture wide, and all the cells should fall into phase. Furthermore by using the chemotaxis system which has an intrinsically differential sensor (rather than an absolute sensor) it is hoped that the oscillations can be driven over many cell cycles.
This project is relatively fundamental but will we hope provide several generally useable and reusable modules for more applied projects where multiple signaling pathways, culture wide oscillators or switches are required. The project involves both experimental constructions, largely derived from previously published work by other authors, resulting in numerous new Biobricks, and also a strong modeling component to understand the function and constraints of the oscillator that we propose to build and the expected effects on bacterial behavior.
Project Details
Serine, a signal molecule
Modified E.coli strains we used for this project are mutated for:
- SdaB gene encoding for a Serine transporter
- SdaC gene encoding for a protein able to degrade Serine into 3-PG.
- CheA gene merged with (SH3)4 : we made sure to get rid of any endogenous and exogenous wild type CheA that could not be able to interact with CusR-LZa or phosphorylated CusR-LZa , leading to a decrease in CheA-SH3 intracellular concentration.
- SerA gene: mutated SerA cannot enter the cell anymore to follow its degradation pathway, leading to a continuous increase of the extracellular Serine concentration. Mutated SerA conserves its enzymatic function.
SerA is controlled by CusR promoter which is activated by the variation of extracellular Serine concentration.
Here, the aim is to synthetize the Serine without interruption. When intracellular Serine concentration is high, the protein goes to the extracellular milieu.
The evolution of Serine concentration stimulates or inhibits the cellular division in our system.
CheA‐SH3, convoluted chemotaxy mecanism
Here, a change in the extracellular Serine concentration is detected by Tsr receptor leading to its activation. Then Tsr inhibits the constitutive synthesis of CheA (Histidine kinase). Consequently, CusR transcription factor cannot be activated by phosphorylation anymore.
When extracellular Serine concentration is stabilized, Tsr is not stimulated anymore and stop inhibiting CheA. As a consequence, CheA becomes activated again and phosphorylates CusR.
Note that CheA-(SH3)4 and CusR-LZa are fusion proteins. SH3-pep-LZa is the linker between the two protein complexes.
Regulation of ppGpp intracellular concentration; green light, go !
In the end, the CusR transcription factor activates RelA-RFP expression. RelA is a factor implicated in the increase of ppGpp intracellular concentration. Thus ppGpp blocks the cellular cycle in S phase, leading to the stoppage of E.coli cellular division. In the mean time, CusR fixes to a specific site near from Mesh1 constitutive promoter leading to the inhibition of Mesh1. At this point, bacteria are in the stationary phase, and will turn red.
As we said before, when extracellular Serine concentration increases, CusR won’t be activated. Consequently, RelA-GFP gene won’t be expressed whereas Mesh1-GFP fusion proteins will be produced. The degradation of ppGpp factor by Mesh1 leads to a decrease of ppGpp intracellular concentration resulting in the resumption of E.coli cellular division. I this case, bacteria will appear green.
As the changes in Serine concentration happens in the whole cellular environment, we’re able then to observe the evolution of the cellular division for all of the bacteria.
Furthermore, using both chemotaxy mechanism and changes in the Serine extracellular concentration we would be able to synchronize cells more efficiently and for a longer period than physical or chemical technics that already exist nowadays.
References
Pertinent articles related to our project:
Top Papers
- Gudipaty SA1, Larsen AS, Rensing C, McEvoy MM. Regulation of Cu(I)/Ag(I) efflux genes in Escherichia coli by the sensor kinase CusS. FEMS Microbiol Lett. 2012 May;330(1):30-7.
- Rudd KE, Bochner BR, Cashel M, Roth JR. Mutations in the spoT gene of Salmonella typhimurium: effects on his operon expression. J Bacteriol. ;163(2):534-42, 1985.
- Whitaker WR1, Davis SA, Arkin AP, Dueber JE. Engineering robust control of two-component system phosphotransfer using modular scaffolds. Proc Natl Acad Sci U S A. 2012 Oct 30;109(44):18090-5.
- Ferullo DJ, Cooper DL, Moore HR, Lovett ST. Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication. Methods. 2009 May;48(1):8-13.
- Peters-Wendisch P1, Stolz M, Etterich H, Kennerknecht N, Sahm H, Eggeling L. Metabolic engineering of Corynebacterium glutamicum for L-serine production. Appl Environ Microbiol. 2005 Nov;71(11):7139-44.
Related Papers
- Wahl A, My L, Dumoulin R, Sturgis JN, Bouveret E.Antagonistic regulation of dgkA and plsB genes of phospholipid synthesis by multiple stress responses in Escherichia coli. Mol Microbiol. 80(5):1260-75, 2011.
- Collins CH. Cell-cell communication special issue. ACS Synth Biol. 2014 Apr 18;3(4):1978.
- Hsiao V, de Los Santos EL, Whitaker WR, Dueber JE, Murray RM. Design and Implementation of a Biomolecular Concentration Tracker. ACS Synth Biol. 2014 May 15.
- Park JH, Oh JE, Lee KH, Kim JY, Lee SY. Rational design of Escherichia coli for L-isoleucine production. ACS Synth Biol. 2012 Nov 16;1(11):532-40.
- Tan MH1, Kozdon JB, Shen X, Shapiro L, McAdams HH. An essential transcription factor, SciP, enhances robustness of Caulobacter cell cycle regulation. Proc Natl Acad Sci U S A. 2010 Nov 2;107(44):18985-90.
- Park H, Saha SK, Inouye M. Two-domain reconstitution of a functional protein histidine kinase. Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6728-32.
- Porter SL, Wadhams GH, Armitage JP. Signal processing in complex chemotaxis pathways.Nat Rev Microbiol. 2011 Mar;9(3):153-65.
- Olson EJ, Hartsough LA, Landry BP, Shroff R, Tabor JJ. Characterizing bacterial gene circuit dynamics with optically programmed gene expression signals. Nat Methods. 2014 Apr;11(4):449-55.
- Skerker JM1, Perchuk BS, Siryaporn A, Lubin EA, Ashenberg O, Goulian M, Laub MT. Rewiring the specificity of two-component signal transduction systems. Cell. 2008 Jun 13;133(6):1043-54.
- Skerker JM1, Prasol MS, Perchuk BS, Biondi EG, Laub MT. Two-component signal transduction pathways regulating growth and cell cycle progression in a bacterium: a system-level analysis. PLoS Biol. 2005 Oct;3(10):e334.
- Gonzalez D, Collier J. Effects of (p)ppGpp on the progression of the cell cycle of Caulobacter crescentus. J Bacteriol. 2014 Jul;196(14):2514-25.
- Thanbichler M. Synchronization of chromosome dynamics and cell division in bacteria. Cold Spring Harb Perspect Biol. 2010 Jan;2(1):a000331.
- Wang X, Vallurupalli P, Vu A, Lee K, Sun S, Bai WJ, Wu C, Zhou H, Shea JE, Kay LE, Dahlquist FW. The linker between the dimerization and catalytic domains of the CheA histidine kinase propagates changes in structure and dynamics that are important for enzymatic activity. Biochemistry. 2014 Feb 11;53(5):855-61.
- Eggeling L, Sahm H. New ubiquitous translocators: amino acid export by Corynebacterium glutamicum and Escherichia coli. Arch Microbiol. 2003 Sep;180(3):155-60.
- Laub MT, Goulian M. Specificity in two-component signal transduction pathways. Annu Rev Genet. 2007;41:121-45.
- Alon U, Camarena L, Surette MG, Aguera y Arcas B, Liu Y, Leibler S, Stock JB. Response regulator output in bacterial chemotaxis. EMBO J. 1998 Aug 3;17(15):4238-48.
- Chang YC, Armitage JP, Papachristodoulou A, Wadhams GH. A single phosphatase can convert a robust step response into a graded, tunable or adaptive response. Microbiology. 2013 Jul;159(Pt 7):1276-85.
- Ganesh I, Ravikumar S, Lee SH, Park SJ, Hong SH. Engineered fumarate sensing Escherichia coli based on novel chimeric two-component system. J Biotechnol. 2013 Dec;168(4):560-6.
- Jonas K. To divide or not to divide: control of the bacterial cell cycle by environmental cues. Curr Opin Microbiol. 2014 Apr;18:54-60.
- Wahl A, My L, Dumoulin R, Sturgis JN, Bouveret E.Antagonistic regulation of dgkA and plsB genes of phospholipid synthesis by multiple stress responses in Escherichia coli. Mol Microbiol. 80(5):1260-75, 2011.
- Collins CH. Cell-cell communication special issue. ACS Synth Biol. 2014 Apr 18;3(4):1978.
- Hsiao V, de Los Santos EL, Whitaker WR, Dueber JE, Murray RM. Design and Implementation of a Biomolecular Concentration Tracker. ACS Synth Biol. 2014 May 15.
- Park JH, Oh JE, Lee KH, Kim JY, Lee SY. Rational design of Escherichia coli for L-isoleucine production. ACS Synth Biol. 2012 Nov 16;1(11):532-40.
- Tan MH1, Kozdon JB, Shen X, Shapiro L, McAdams HH. An essential transcription factor, SciP, enhances robustness of Caulobacter cell cycle regulation. Proc Natl Acad Sci U S A. 2010 Nov 2;107(44):18985-90.
- Park H, Saha SK, Inouye M. Two-domain reconstitution of a functional protein histidine kinase. Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6728-32.
- Porter SL, Wadhams GH, Armitage JP. Signal processing in complex chemotaxis pathways.Nat Rev Microbiol. 2011 Mar;9(3):153-65.
- Olson EJ, Hartsough LA, Landry BP, Shroff R, Tabor JJ. Characterizing bacterial gene circuit dynamics with optically programmed gene expression signals. Nat Methods. 2014 Apr;11(4):449-55.
- Skerker JM1, Perchuk BS, Siryaporn A, Lubin EA, Ashenberg O, Goulian M, Laub MT. Rewiring the specificity of two-component signal transduction systems. Cell. 2008 Jun 13;133(6):1043-54.
- Skerker JM1, Prasol MS, Perchuk BS, Biondi EG, Laub MT. Two-component signal transduction pathways regulating growth and cell cycle progression in a bacterium: a system-level analysis. PLoS Biol. 2005 Oct;3(10):e334.
- Gonzalez D, Collier J. Effects of (p)ppGpp on the progression of the cell cycle of Caulobacter crescentus. J Bacteriol. 2014 Jul;196(14):2514-25.
- Thanbichler M. Synchronization of chromosome dynamics and cell division in bacteria. Cold Spring Harb Perspect Biol. 2010 Jan;2(1):a000331.
- Wang X, Vallurupalli P, Vu A, Lee K, Sun S, Bai WJ, Wu C, Zhou H, Shea JE, Kay LE, Dahlquist FW. The linker between the dimerization and catalytic domains of the CheA histidine kinase propagates changes in structure and dynamics that are important for enzymatic activity. Biochemistry. 2014 Feb 11;53(5):855-61.
- Eggeling L, Sahm H. New ubiquitous translocators: amino acid export by Corynebacterium glutamicum and Escherichia coli. Arch Microbiol. 2003 Sep;180(3):155-60.
- Laub MT, Goulian M. Specificity in two-component signal transduction pathways. Annu Rev Genet. 2007;41:121-45.
- Alon U, Camarena L, Surette MG, Aguera y Arcas B, Liu Y, Leibler S, Stock JB. Response regulator output in bacterial chemotaxis. EMBO J. 1998 Aug 3;17(15):4238-48.
- Chang YC, Armitage JP, Papachristodoulou A, Wadhams GH. A single phosphatase can convert a robust step response into a graded, tunable or adaptive response. Microbiology. 2013 Jul;159(Pt 7):1276-85.
- Ganesh I, Ravikumar S, Lee SH, Park SJ, Hong SH. Engineered fumarate sensing Escherichia coli based on novel chimeric two-component system. J Biotechnol. 2013 Dec;168(4):560-6.
- Jonas K. To divide or not to divide: control of the bacterial cell cycle by environmental cues. Curr Opin Microbiol. 2014 Apr;18:54-60.
Protocols
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Results and Parts validation
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Conclusions
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