Part:BBa_K5201002

Characterisation of BBa_K5201002: HongKong-UCCKE

Characterization

Colony PCR and Agarose gel electrophoresis (AGE)

Fig. 11 Agarose gel electrophoresis of the colony PCR products of composite part (BBa_K5201002)

In our project, we used E. coli DH5α as host. Within its plasmid backbone, we inserted Lac Promoter (BBa_R0010), pmHAS*1-703 (BBa_K5201000), RBS (BBa_B0030), kfiD (BBa_K5201001) and a double terminator (BBa_B0015) into our gene circuit. Lac Promoter is chosen since it is an inducible promoter where it can be induced by lactose or isopropylthio-galactoside (IPTG). In order for stronger induction, a strong RBS (BBa_B0030) is chosen. Most importantly, we have inserted pmHAS*1-703 and kfiD gene to produce HA. pmHAS*1-703 encodes a class II hyaluronic acid synthase (HAS) which converts UDP-glucuronic acid and UDP-N-Acetylglucosamine into HA. The residues 1-703 and 704-972 encode the catalytic domain and the transmembrane domain respectively. By using only the first 703 amino acids, we can express a non-membrane bound but functional HAS. Meanwhile, kfiD is responsible for synthesis of UDP-glucose-6-dehydrogenase (UGDH) which converts UDP-glucose into UDP-glucuronic acid. This allows the transformed bacteria to synthesize HA more efficiently.