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.

Strains used in the project

• Escherichia coli W3110 was used for chassis construction.
• Escherichia coli DH5alpha and TG1 strains were used for regular cloning.

Strain storage

For long-term conservation, 750 μL of an exponentially grown strain were mixed with 250 μL of glycerol 80%, and at -80°c in a cryotube.

Culture medium

• Regular bacterial growth was performed in LB medium or LB-agar plates (DIFCO) supplemented with the appropriated antibiotics if necessary: ampicillin 100 μM, kanamycin 50 μM, chloramphenicol 30 μM. When required, IPTG (Isopropyl β-D-1-thiogalactopyranoside ) was added to the final concentration of 500 μM to induce gene expression from Plac promoters.

  Growth was conducted at 37°C for regular experiments, and at 30°C when carrying temperature sensitive plasmids.

• The SMG medium used to characterize the BBa_K1349001 part was prepared based on the work of (Rudd et al.,1985).

  SMG plate composition:

  M9 salts	1X
  MgSO4	1mM
  CaCl2	0.1mM
  VitB1	0.5µg/ml
  Glucose	0.2%
  Serine	1mM
  Methionine	1mM
  Glycine	1mM
  Bactoagar	15g/L

References:

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.

Plasmid extraction

Performed with the Macherey-Nagel Kit, following the manufacturer instructions.

PCR clean-up

Performed with the Promega Kit, following the manufacturer instructions.

Competent cells

Cells were grown in LB medium as a starter O/N. The day after, the culture was back diluted in 100 mL of LB to reach an OD600=0.05.

When the culture reached and OD600 of 0.4 to 0.6, cells were centrifuged at 5000 rpm during 10 min. The cell pellet was carefully re-suspended in 1/2V of CaCl2 50 mM at 4°C. After an incubation of 10 min on ice, the cells were pelleted by centrifugation. The pellet was re-suspended in 1/20V of CaCl2 50 mM, Glycerol 15%. Aliquots of competent cells were stored at -80°C.

Transformation

Performed following the protocol published on the iGEM website : here

Lambda Red mutant construction

This protocol was adapted from the initial protocol published by Datsenko and Wanner (2000).

Reference:
Datsenko KA1, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000 6;97(12):6640-5.

• Day 1:

  • Transform W3110 competent cells with the pKOBEG plasmid (cmR). Grow at 30°C o/n
  • PCR-amplify the FRT-kan-FRT cassette from the pKD4 plasmid (AmpR) using the set of primers targeting the gene to be deleted. Check the PCR product on an agarose gel. Then digest the pKD4 matrix plasmid with 1uL DpnI enzyme, and incubate at 37°C during 2 hrs. Clean-up the PCR product with the promega kit.

• Day 2:

  From one isolated W3110 pKOBEG clone, grow the cells at 30°C. When the culture reach an OD of 2.2, add arabinose to a final concentration of 0.05% and incubate 2 hrs at 30°C or until OD600=1. Switch the temperature to 42°C to get rid off the pKOBEG plasmid. Centrifuge the culture at 4°C and prepare a stock of electrocompetent cells.

  Electroporate 10 ul of purified PCR product into 50 ul of electrocompetent cells. Grow the transformed cells on LB+kan 50uM plates, and incubate at 37°C o/n.

• Day 3:

  Restreak 25 clones on LB+Kan plates and Lb+Cm plates, incubate at 37°C O/N.

• Day 4:

  Select 8 clones resistant to Kan but sensitive to Cm. Check the correct replacement of the targeted gene by the FRT-Kan-FRT cassette with primers exterior to the gene.

• Day 5:

  Cultivate and freeze 2 independent clones.

To flip the FRT-KAN-FRT cassette

• Day 6:

  Prepare a stock of electrocompetent W3110 mutant.

  Electroporate the pCP20 plasmid (CmR, ApR) encoding the flipase and spread the cells on LB+Ap+Cm. Incubate at 30°C O/N

• Day 7:

  To prepare a culture starter, inoculate 1 clone in LB at 30°c O/N

• Day 8:

  Back-dilute the culture 100x in LB without antibiotics. Grow at 30°c until you reach an OD600=0.3. Switch the temperature to 37°C until OD600= 0.8. Back-dilute the culture 100x in LB for 5 hrs. Spread 10 ul of the culture in LB plate without antibiotics to get isolated colonies.

• Day 9:

  Select 20 isolated clones and re-patch on LB, LB+Kan, LB+AP+CM.

• Day 10:

  Select 8 clones sensitive to Kan, Ap and Cm. Check by colony-PCR that the FRT-KAN-FRT cassette was truly deleted.

SLIC cloning

One-step sequence- and ligation-independent cloning (SLIC) was performed following the protocol published as supplemental data in the original manuscript of Jeong and collaborators. This protocol can be found here

References
Jeong JY1, Yim HS, Ryu JY, Lee HS, Lee JH, Seen DS, Kang SG. One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies. Appl Environ Microbiol. 2012 ; 78(15):5440-3.

3A cloning

Performed following the protocol published on the iGEM website : here

Result 1: validation of the RelA part: BBa_K1349001

The relA gene encodes a ppGpp synthetase. In bacteria, ppGpp (guanosine 3'-diphosphate 5-' diphosphate) acts as a signaling molecule that regulates a variety of cellular metabolisms in response to changes in the nutritional state of the cells.

This part was designed to allow the rapid synthesis of the alarmone ppGpp, responsible for the stringent response in E. coli. In the context of our project ppGpp is accumulation used to stop initiation of cell division.

How we tested the BBa_K1349001 part:

Expression of the BBa_K1349001 part is expected to cause accumulation of ppGpp and so reduce growth rate of a wild-type cell. At the opposite, expression of this part in a MG1655∆relA mutant unable to make ppGpp is expected to complement the mutant.

As seen on the figure below, our part is functional as it is able to fully complement the MG1655∆relA mutant. See lab-book for details on the experimental procedure.

Attribution of the figure: Dr. Emmanuelle Bouveret.