Team:Grenoble/Biology/AND gate


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iGEM Grenoble 2012



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].

In presence of L-arabinose and the CRP-cAMP complex, the promoter is activated thus enabling the transcription of the downstream elements.

This AND gate provides a filter to biological noise. Check out our main results page to for the AND gate characterization.

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