Composite

Part:BBa_K2963021

Designed by: Experiment group members of JNU-China   Group: iGEM19_JNU-China   (2019-09-12)
Revision as of 05:00, 19 October 2021 by Wubeizhongxinghua (Talk | contribs)


pgsBCA- encoding a poly-γ-glutamic acid synthetase

PgsBCA complex, consisting of three subunits, is composed of PgsB、PgsC and PgsA. PgsB catalyzes poly-γ-glutamic acid synthesis．And PgsC links PgsB and PgsA in the membrane. While PgsA transports poly-γ-glutamic acid outside the cell. In our project, we use the PgsBCA complex to biosynthesize poly-γ-glutamic acid.

Usage and Biology

The BCA genes from Bacillus sp. encode a γ-PGA synthetase located on the cell membrane which is capable of polymerizing glutamic acid to form poly-γ-glutamic acid. In Bacillus subtilis, BCA are called pgsBCA. This part is used producing L-glutamate-rich γ-PGA.

Characterization

We used NMR to detect γ-PGA and HPLC to analyze L- glutamate ratio of γ-PGA. The results show we have successfully produced L-glutamate-rich γ-PGA.

NMR.png

By the NMR detection of the fermentation product, the specific hydrogen peaks a, b, and c on the γ-amide bond on the γ-polyglutamic acid could be detected at the corresponding time points. It is indicated that the synthetase pgsBCA genes of Bacillus subtilis heterologously expressed in Corynebacterium glutamicum, and the target product γ-polyglutamic acid is successfully produced.

HPLC.png

We used HPLC to detect the L-glutamate monomer ratio of the γ-PGA we have produced. The results show that the L-glutamic acid monomer ratio reaches more than 90%. This part is working and we have produced L-glutamate-rich γ-PGA.

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.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 2207
    Illegal PstI site found at 2188
    Illegal PstI site found at 3467
    Illegal PstI site found at 3753
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2365
    Illegal NheI site found at 2963
    Illegal NheI site found at 4254
    Illegal PstI site found at 2188
    Illegal PstI site found at 3467
    Illegal PstI site found at 3753
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 2207
    Illegal PstI site found at 2188
    Illegal PstI site found at 3467
    Illegal PstI site found at 3753
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 2207
    Illegal PstI site found at 2188
    Illegal PstI site found at 3467
    Illegal PstI site found at 3753
    Illegal NgoMIV site found at 2586
    Illegal AgeI site found at 4111
  • 1000
    COMPATIBLE WITH RFC[1000]


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