This DNA biobrick was designed in order to produce C8-CAI-1 of Vibrio harveyi in E. coli strain.
1- Biological background
The production of the Vibrio harveyi quorum sensing inducer (C8-CAI-1) is under the control of the cqsA gene coding for the CqsA synthase. This enzyme catalyzes the production of C8-CAI-1, an analog of CAI-1 quorum sensing inducer from Vibrio cholerae. The CqsA catalyzes the following reaction :
2- Usage in iGEM projects
The BBa_K2278001 cames from the sensing module of the Croc’n cholera project (team INSA-UPS-France 2017)
It was designed to produce C8-CAI-1 to simulate the presence of Vibrio cholerae in a water sample and so, to allow the validation of our Vibrio cholerae quorum sensing based detection system.
The part includes Vibrio harveyi cqsA synthase under the control of an IPTG inducible promoter.
The C8-CAI-1 producing system is inducible in order to avoid toxicity problems and high metabolic activity during cells growth.
As the cqsA gene comes from Vibrio harveyi which is a BSL1 organism, it can be used as substitute in experiment about the Vibrio cholerae quorum sensing in BSL1 condition.
Characterization
1- Molecular biology
[Lien vers le wiki part]
VhCqsA, the enzyme coding from the C8-CAI-1 producer (CqsA) has been cloned with success and sequenced.
2- Validation of the production of C8-CAI-1 and its structure
We use MS and NMR analysis approaches to show that we are able to produce C8-CAI-1 using E.coli
MS spectra :
[Image MS avec le pic qui correspond à la masse de C8-CAI-1 comparé a coli plasm vide).