Part:BBa_K5201002
pLac-B0010-pmHAS*1-703-kfiD-B0015
Characterization Colony PCR and Agarose gel electrophoresis (AGE)
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, a non-membrane bound but functional HAS is produced. 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 957
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2592
Illegal BamHI site found at 3124
Illegal XhoI site found at 3467 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1456
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