Part:BBa_K2808000
N-acetyl glucosamine-binding protein A (gbpA) gene for the detection of Vibrio cholera
The gbpA gene in Vibrio cholerae codes for the N-acetyl glucosamine-binding protein A (GbpA), which is a chitin-binding protein involved in the attachment of V. cholerae to environmental chitin surfaces and human intestinal cells. The gbpA gene has been found to be consistently present and highly conserved in V. cholerae. GbpA has been used as a target gene for the species-specific detection of V. cholerae O1, O139, Non-O1/Non-O139.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1128
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Improvement upon part BBa_K2299000
Instead of the 39 bp sequence found in CTX RstA gene that is used by BBa_K2299000, part BBa_K2808000 uses gbpA gene for the detection of V. cholera. The advantages of using gbpA as a gene target compared to ctxA are various such as ctxA is involved in the production of the cholera toxin production which requires special safety precautions during bacterial transformation, lab experiments, shipping reagents, customs, etc. whereas the use of gbpA solves these problems as gbpA is not directly involved in the production of cholera toxin.
Experimental Validation
Throughout our project, we successfully ligated our gene gbpA to the pSB1CR linearized backbone and transformed it into competent DH5-alpha E. coli cells. The part was validated by running a double restriction digestion reaction and PCR on the gene fragment using the respective PCR primers and the amplified regions were obtained as expected. Appropriate bands were seen in the respective lanes, which indicated that the part was successfully ligated and transformed.
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Source
Vibrio cholera
References
Vezzulli L, et al. (2015) gbpA as a novel qPCR target for the species-specific detection of Vibrio cholerae O1, O139, non-O1/non-O139 in environmental, stool, and historical continuous plankton recorder samples. PloS one 10(4):e0123983.
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