Part:BBa_K2278002
Vibrio cholerae CAI-1 (quorum sensing inducer) generator
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 619
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Introduction
This DNA biobrick was designed in order to produce CAI-1 of Vibrio cholerae in E. coli strain.
1- Biological background
The production of the Vibrio cholerae quorum sensing inducer (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_K2278002 cames from the sensing module of the Croc’n cholera project (team INSA-UPS-France 2017) It was designed to produce CAI-1 by an E.coli 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 cholerae cqsA synthase under the control of an IPTG inducible promoter. The 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 cholerae which is a BSL2 organism, it has to be manipulated under safe condition even if this gene is not directly involved in pathogenicity or antibiotic resistance.
To avoid safety issues, we recommand to use BBa_K2278001 which produce a quorum sensing analog molecule : C8-CAI-1
Experiments
Molecular biology
The cqsA coding gene was placed in silico under the control of the plac promoter (BBa_R0040), a strong RBS (BBa_B0034) and a terminator (BBa_B1006). IDT performed the DNA synthesis and delivered the part as gBlock. The construct was cloned by conventional ligation into pSB1C3 plasmid and then transformed into E. coli Dh5 alpha strain. Three transformants were obtained.
Analysis of the restriction mapPerceptives:
The biobrick can be validated by a bioluminescence assay with V. harveyi JMH626 with deleted cqsA.Design Notes
Terminator : BBa_B1006* is mutated substitution at the 35th pair (initial A->T) due to IDT complexity requirements for gBlocks synthesis. The effect of the mutation was not investigated.
This biobrick is very close to Part:BBa_K356001. We proposed an alternative design by inversing the RBS and promoter place in the initial design. The promoter is now before the RBS and we used an inducible promoter in order to avoid toxicity problems and high metabolism activity during cell growth.
Source
This enzyme cames from Vibrio cholerae DNA genomic sequence.
References
<p>Ng W-L, Perez LJ, Wei Y, Kraml C, Semmelhack MF & Bassler BL (2011) Signal production and detection specificity in Vibrio CqsA/CqsS quorum-sensing systems: Vibrio quorum-sensing systems. Molecular Microbiology 79 1407–1417
https://www.ncbi.nlm.nih.gov/pubmed/21219472Wei, Y., Perez, L., Ng, W., Semmelhack, M. and Bassler, B. (2011). Mechanism of Vibrio cholerae Autoinducer-1 Biosynthesis. ACS Chemical Biology, 6(4), pp.356-365.
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