Part:BBa_K2963009:Design
capB*CA - encoding poly-γ-glutamic acid synthetase
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1912
Illegal EcoRI site found at 2756
Illegal EcoRI site found at 2815 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1912
Illegal EcoRI site found at 2756
Illegal EcoRI site found at 2815
Illegal NheI site found at 2365
Illegal NheI site found at 2963
Illegal NheI site found at 4281 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1912
Illegal EcoRI site found at 2756
Illegal EcoRI site found at 2815 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1912
Illegal EcoRI site found at 2756
Illegal EcoRI site found at 2815 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1912
Illegal EcoRI site found at 2756
Illegal EcoRI site found at 2815
Illegal NgoMIV site found at 2590 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
Our team cloned the capB*CA genes (B* is a mutant of B) We used ePathBrick loop vector pZM1 (Ptac) to modularize B*CA genes and assembled them into pZM1 vector as the following structure:
We assembled this part into PZM1 plasmid which contains a regulatory gene (lacI) to construct our gene circuit. The gene circuit need to be induced by IPTG to initiate expression of the polymerase genes to synthesize our product L-glutamate-rich γ-PGA. We transferred this gene circuit into our chassis microorganism: Corynebacterium glutamicum for fermentation experiments, and verified the fermentation products by NMR. The L- glutamate ratio of γ-PGA was measured by HPLC, and the result showed that the content of L-glutamic acid in γ-PGA reached over 90%. We have successfully produced L-glutamate-rich γ-PGA. If you want to know more details and related experiments about this part, we recommend you to visit our experiment page.
Source
Bacillus Genome.
References
1. Xu P, Vansiri A, Bhan N, et al. ePathBrick: a synthetic biology platform for engineering metabolic pathways in E. coli[J]. ACS Synthetic Biology, 2012, 1(7): 256-266.
2. Peng Yingyun. Study on the production, synthesis mechanism and antifreeze of γ-polyglutamic acid. Diss. Jiangnan University, 2015.
3. Sung M H, Park C, Kim C J, et al. Natural and edible biopolymer poly-gamma-glutamic acid: synthesis, production, and applications [J]. Chemical Record, 2005, 5(6): 352-366.