Team:WashU StLouis/Improve

We decided to improve the existing part, BBa_K1021001, which is the coding sequence for Neomycin Phosphotransferase (nptII). Neomycin phosphotransferase is an enzyme that is known to confer resistance to several antibiotics such as neomycin, kanamycin, geneticin, and paramomycin. The original creators of the part intended to use it in the selection of the fungus, C. heterosporus, and the moss, P. Patens. There didn't seem to be a codon-optimized option for DH5α E. coli which is commonly used in biological research labs. In addition, kanamycin is a common antibiotic used for selection as well.

In order to develop a better option for DH5α E. coli cells, we used the IDT codon optimizer to codon optimize the sequence from the original part (BBa_K1021001). After receiving the DNA, we selected a medium promoter and a medium RBS from a part (BBa_K608006) already in the registry. We ligated the two sequences together in a pSB1C3 plasmid and transformed it into DH5α E. coli cells, plating them both on a chloramphenicol plate and a kanamycin plate. Colonies appeared on both plates, supporting that the gene works. Below is a picture of the kanamycin plate.