Part:BBa_K3866015

PhaA-RBS-PhaB-RBS-Crt-RBS-Ter GB compatible with B3-B5

The production of butyrate by bacteria can be possible with a set of genes. The superpart 1 includes only four of the nine genes that catalyze the formation of butyric acid. Those are PhaA, PhaB, Crt and Ter genes.

Fig.2:J23115 with RBS

Usage and Biology

First, PhaA and BktB are two β-ketothiolases that carry out the homo-fusion of two acetyl-CoA moieties into acetoacetyl-CoA. Then, the acetoacetyl-CoA is reduced to (S)-3-hydroxybutyryl-CoA (3-HB-CoA) by NADH-dependent Hbd or (R)-3-HB-CoA by NADPH-dependent PhaB. Both (R)-3-HB-CoA and (S)- 3-HB-CoA can be converted to (R)-3-HB and (S)-3-HB, respectively, by a CoA-removing enzyme, such as Pct, TesB, or Ptb/Buk. Alternatively, (R)-3-HB-CoA and (S)-3-HB-CoA can be converted to crotonyl-CoA by PhaJ and Crt, respectively. Crotonyl-CoA is then reduced to butyryl-CoA by NADH-dependent Ter, and subsequently converted to butyrate by one of the above CoA-removing enzymes.

Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in pUPD2 BBa_K3505007 and has overhangs compatible for GoldenBraid cloning. The Promoter has position B3-B5.

Fig.1:The overhangs of this part in the GoldenBraid Grammar

Verification and Cloning

Digestion with EcoRV Expected bands at:

• 4807 bps
• 1262 bps

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 357
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 357
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3315
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 357
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 357
  Illegal NgoMIV site found at 122
  Illegal NgoMIV site found at 226
  
• 1000
  COMPATIBLE WITH RFC[1000]

Source

Synthesized by Twist Biosciences.

References

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