Biological "AND" gate

Promoter paraBAD

    

cyclic Adenosine MonoPhosphate

Gene transcription

A new characterization of the paraBAD promoter

• M9 complement with 0.03% of glucose and 0.03% of acetate (1)
• M9 complement with 0.1% of glycerol (2)
• M9 complement with 0.1% of acetate (3)

medium (1)

medium (2)

medium (3)

In a first place, we tested the robustness of the AND gate.

medium (1)

medium (2)

medium (3)

As you can see on this figure even after few hours there is no fluorescent expression if one activator is absent.
Now for our system we studied the half-expression of GFP versus the cAMP concentration at 1.6% of arabinose.

medium (1)

medium (2)

medium (3)

As you can see in the two first media we have the half expression of GFP at 0.4mM of cAMP whereas in the third medium its concentration is: 0.8mM. We want a sensitive detector, therefore the third medium conditions are not optimum.

medium (1)

medium (2)

medium (3)

One of ours imperative is : a fast response. Consequently, we need to know the time it takes to reach the half expression.

medium (1)

medium (3)

Conclusion

With those different experiments we demonstrated that paraBAD works as expected. It will be activated only if there is arabinose and cAMP in the medium. It appears that both cAMP concentration and time response required for a half expression of GFP depends on the medium. As we want a quick response as well as a satisfying sensivity, these analysis shows that the best medium for our device to come would be the M9 medium complemented with 0.1% of glycerol (second medium).

References

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• [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