Difference between revisions of "Part:BBa K3866015"

Latest revision as of 19:29, 26 September 2021

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:PhaA, PhaB, Crt, Ter genes constructed in an operon in a PUPD2 vector, with compatible ends for Golden Braid Cloning

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

Fig.2:PhaA,PhaB,Crt,Ter genes into a PUPD2 vector. Digestion with EcoRV Expected bands at: *4807 bps *1262 bps

Sequence and Features

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.