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Team:Grenoble/Biology/AND gate
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paraBAD
In order to develop our device we needed a biological AND gate. We found a promoter which can be activated by 2 molecules : CRP-cAMP complex and the AraC protein. The paraBAD promoter has two states; in absence of L-arabinose the paraBAD promoter is repressed by AraC, whereas the the paraC promoter is activated (unless an excess of AraC is present) [Schemes].
Cyclic Adenosine Monophosphate
In order to make our AND work we need to produce cyclic Adenosine Monophosphate (cAMP). cAMP is produce by adenyl cyclase (encoded by the cyaA gene). It is an enzyme which catalyses the conversion of ATP to 3’,5’-cAMP. In Escherichia coli, cAMP is involved in carbon catabolite repression [1] and binds to the cAMP receptor protein (CRP). The corresponding complex (CRP-cAMP) is a transcriptional factor controlling the expression of more than 220 operons [2]. It has been known for a long time that E. coli actively exports cAMP into the growth medium [3][4].Gene transcription
The transcription of the cyaA gene and the crp gene is negatively regulated by CRP-cAMP [5] [6]. The translation of adenylate cyclase mRNA is ineffective as to prevent excessive synthesis of adenylate cyclase. This can be attributed to the fact that overproduction of cAMP is lethal to Escherichia coli possibly due to an accumulation of methylglyoxal [7] [8]. As we want to use paraBAD like an AND Gate and because high a concentration of cAMP is lethal for E. Coli we make our device in a BW25113 ΔcyaA.References
- [1] Gorke, B., & Stulke, J. 2008. Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nat Rev Microbiol, 6(8), 613–24.
- [2] Keseler, I. M., Bonavides-Martinez, C., Collado-Vides, J., Gama-Castro, S., Gunsalus, R. P., Johnson, D. A., Krummenacker, M., Nolan, L. M., Paley, S., Paulsen, I. T., Peralta-Gil, M., Santos-Zavaleta, A., Shearer, A. G., & Karp, P. D. 2009. EcoCyc: a comprehensive view of Escherichia coli biology. Nucleic Acids Res, 37(Database issue), D464–70.
- [3] Goldenbaum, P. E., & Hall, G. A. 1979. Transport of cyclic adenosine 3’,5’-monophosphate across Escherichia coli vesicle membranes. J Bacteriol, 140(2), 459–67.
- [4] Makman, R. S., & Sutherland, E. W. 1965. Adenosine 3’,5’-Phosphate in Escherichia Coli. J Biol Chem, 240, 1309–14.
- [5] K Mori and H Aiba
- [6] Cell. 1983 Jan;32(1):141-9. Autoregulation of the Escherichia coli crp gene: CRP is a transcriptional repressor for its own gene. Aiba H
- [7] Rollie S. Ackerman, Nicholas R. Cozzarelli and Wolfgang Epstein.Accumulation of Toxic Concentrations of Methylglyoxal by Wild-Type Escherichia coli K-12.J. Bacteriol. August 1974 vol. 119 no. 2 357-362
- [8] Jan Weber†, Anke Kayser‡ and Ursula Rinas. Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. II. Dynamic response to famine and feast, activation of the methylglyoxal pathway and oscillatory behaviour. Microbiology March 2005 vol. 151 no. 3 707-716
- [Schemes] Université d’Orléans – UFR Sciences & Centre de Biophysique Moléculaire UPR4301
