Part:BBa_K2963021:Design

pgsBCA- encoding a poly-γ-glutamic acid synthetase

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]

Design Notes

Our team cloned the pgsBCA genes, We used pZM1 (Ptac) to modularize BCA 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 results show 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 subtillus.