Composite

Part:BBa_K2963043

Designed by: Experiment group members of JNU-China   Group: iGEM19_JNU-China   (2019-10-18)


Producing γ-PGA with different D/L glutamate monomer ratio(R) (two lacO in Ptac)

Usage and Biology

We used this part to produce different D/L monomer ratios of γ-PGA.

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 licheniformis, BCA are called capBCA. We used the B* gene, a mutant of B gene.

The racE gene is derived from Bacillus subtilis and it encodes a racemase which can converts L-glutamate to D-glutamate.

We assembled the capB*CA genes and racE gene which is under the control of tac promoter containing one lacO together to construct this part using plasmid PZM1.

Characterization

We transferred this part into our chassis microorganism Corynebacterium glutamicum using plasmid PZM1. And we used HPLC to detect the D/L monomer ratio of γ-PGA. The result shows as below:

Picture1 HPLC2.png

Picture1 is the L-glutamate monomer ratio in γ-PGA we have produced using capB*CA genes and the result reaches about over 90%.


Picture2 HPLC3.png

Picture2 is the result of BBa_K2963043. This part contains capB*CA genes and racE gene which is under the control of tac promoter with one operator(lacO). The L-glutamate monomer ratio reaches about 32%.

When we linked the capB*CA with racE gene, the L-glutamate monnmer ratio was changing. This part is working. All the results show that we have achieved the goal of producing different D/L glutamate monomer ratio of γ-PGA preliminary.

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.Mutalik, Vivek K, et al. "Precise and reliable gene expression via standard transcription and translation initiation elements." Nature Methods 10.4(2013):354.

Sequence and Features


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
    Illegal NheI site found at 5279
  • 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]


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