Composite

Part:BBa_K3866021

Designed by: Ioanna Gkoni   Group: iGEM21_Thessaly   (2021-09-23)
Revision as of 10:48, 29 September 2021 by Igkoni (Talk | contribs) (→‎References)


ParaBAD:RBS-Superpart 1-double terminator

The production of butyrate by bacteria can be possible with a set of genes. This part includes the promoter ParaBAD with RBS(baba), the superpart 1(baba) and the double terminator(baba). The superpart 1 has only four of the nine genes that catalyze the formation of butyric acid. Those are PhaA, PhaB, Crt and Ter genes.

Fig.2:The alpha2 vector that includes the transcriptional unit of the Operon 1 with the ParaBAD promoter and a double terminator. The operon 1 consists of the PhaA, PhaB, Crt and Ter genes, responsible for the butyrate production.

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 alpha 2 vector BBa_K3505009 and has overhangs compatible for GoldenBraid cloning.



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1608
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1608
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4566
    Illegal BamHI site found at 1148
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1608
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1608
    Illegal NgoMIV site found at 1373
    Illegal NgoMIV site found at 1477
    Illegal AgeI site found at 983
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 965

Source

Synthesized by Twist Biosciences.

References

  • Miscevic D, Srirangan K, Kefale T, Abedi D, Moo-Young M, Chou CP. Production of cellulosic butyrate and 3-hydroxybutyrate in engineered Escherichia coli. Appl Microbiol Biotechnol. 2019 Jul;103(13):5215-5230. doi: 10.1007/s00253-019-09815-x. Epub 2019 May 2. PMID: 31049621.

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