Team:NYU Abu Dhabi/Improve

<!DOCTYPE html>

Improve

Improvement upon part BBa_K2299000

The 2018 iGEM NYU Abu Dhabi designed a BioBrick that includes a gene that can be targeted for species-specific detection of Vibrio cholera.

Part BBa_K2808000 was designed by including 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.

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.

gbpA BioBrick double digestion reaction

Figure 1: Agarose gel electrophoresis showing double digestion of the gbpA BioBrick BBA_K2808000 (gbpA gene (1458 bp) inserted into the pSB1C3 backbone (2070 bp)) with EcoRI and PstI. (Lane 1) 500 bp DNA ladder (Bio-Rad); (Lane 2) gbpA BioBrick; (Lane 3) gbpA BioBrick digested with EcoRI and PstI.

BioBricks PCR amplification

Figure 1: Agarose gel electrophoresis showing PCR amplification of: gbpA BioBrick BBA_K2808000; hipO BBA_K2808001; invA BBA_K2808002; lmo0733 BBA_K2808003. (Lane 1) 500 bp DNA ladder (Bio-Rad); (Lane 2) gbpA BioBrick; (Lane 3) negative control for gbpA BioBrick; (Lane 4) hipO BioBrick; (Lane 5) negative control for hipO BioBrick; (Lane 6) invA BioBrick; (Lane 7) negative control for invA BioBrick; (Lane 8) lmo0733 BioBrick; (Lane 9) negative control for lmo0733 BioBrick. Lanes 2, 4, 6, and 8 show bands around 1019 bp, 203 bp, 818 bp, and 403 bp respectively as expected for the PCR products of the gbpA, hipO, invA, and lmo0733 BioBricks respectively.