Difference between revisions of "Part:BBa K2963009:Design"

 
 
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===Design Notes===
 
===Design Notes===
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In order to express PgsBCA complex heterologously in corynebacterium glutamicum, we add Ptac promoter with a short RBS before the start codon and T7 terminater of each protein， respectively.
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Our team cloned the <i>capB*CA</i> genes (<i>B*</i> is a mutant of <i>B</i>) We used ePathBrick loop vector pZM1 (Ptac) to modularize <i>B*CA</i> genes and assembled them into the following structure:
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[[image:CapBCA.png|400px]]
  
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We assembled this part into PZM1 plasmid which contains a regulatory gene (<i>lacI</i>) 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 we verified the fermentation products by NMR. The L- glutamate ratio of γ-PGA was measured by HPLC, and the results show that the content of L-glutamic acid in γ-PGA reaches 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===
 
===Source===
  
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Bacillus Genome.
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<i>Bacillus sp.</i> Genome.
  
 
===References===
 
===References===
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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.
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2. Peng Yingyun. Study on the production, synthesis mechanism and antifreeze of γ-polyglutamic acid. Diss. Jiangnan University, 2015.
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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.

Latest revision as of 12:46, 19 October 2019


capB*CA - encoding poly-γ-glutamic acid synthetase


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1912
    Illegal EcoRI site found at 2756
    Illegal EcoRI site found at 2815
  • 12
    INCOMPATIBLE 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
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1912
    Illegal EcoRI site found at 2756
    Illegal EcoRI site found at 2815
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1912
    Illegal EcoRI site found at 2756
    Illegal EcoRI site found at 2815
  • 25
    INCOMPATIBLE 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
  • 1000
    COMPATIBLE 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 the following structure:

CapBCA.png

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 we verified the fermentation products by NMR. The L- glutamate ratio of γ-PGA was measured by HPLC, and the results show that the content of L-glutamic acid in γ-PGA reaches 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 sp. 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.