Part:BBa_K4808008

thrABC

In Escherichia coli, thrA, thrB and thrC are arranged adjacently on the chromosome to form the thrABC operon. The thrABC operon controls several key enzymes, from aspartate to L-threonine synthesis. Research on thrABC expression and regulation is important for L-threonine synthesis and the downstream products, L-isoleucine and L-glycine. Overexpression of the thrABC operon in a bacterial strain increased L-threonine production significantly.

In our project, we increased the copy number of thrABC in E. coli to increase the production of a-KB.

Characterization

We tried modifying the upstream pathway of Threonine for more a-KB by increasing the copy number of gene thrABC in an effort to enhance the conversion of Aspartate into Threonine (Fig.A). For upstream modification, we used pcr to obtain our gene thrABC from DH5a genome, and then constructed two plasmids, p321-thrABC (constitutive expression) and p15a-thrABC (inducible expression). After the indication of a successful construction of plasmid from DNA sequencing (Fig.c), the two plasmids were each transformed into AIS-2, and after fermentation we measured the a-kb production. Unfortuanatley, the a-kb production did not improve, and instead did the contrary. This may be because, instead of an increase in threonine, an inhibition of the thrABC gene in the E.coli genome was inflicted by the externally transformed thrABC gene that expresses via plasmid, in the end leading to an overall decrease in production of a-kb.

Figure : (A)the pathway of Aspartate to Threonine in E.coli. (B)Obtain gene fragments thrABC and vectors p321 and p15A. (C) sequencing verified the construction of plasmids p321-thrABC and P15A-thrABC. (D) the a-kb production of AIS-2 with plasmid.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 3506
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3506
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3506
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 3506
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 3506
  Illegal NgoMIV site found at 403
  Illegal NgoMIV site found at 3357
  Illegal AgeI site found at 912
  Illegal AgeI site found at 1839
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 3614