Part:BBa_K2260000
PhaCBA operon w/ Histidine tags
Overview
The naturally occuring phaCAB operon in R. eutropha H16 is involved in biosynthesis of poly[(R)-3-hydroxybutyrate] (PHB) ___!!source!!____. It utilizes actyl-coA, which is a product of the glycolysis pathway ___!!source!!____. Transcription of the phaCAB operon leads to expression of the following enzymes in the order: pha synthase, acetoacetyl-CoA reductase, and 3-ketothiolase. The expression of phaA leads to expression of 3-ketothiolase that converts acetyl-coA to acetoacetyl-CoA. The acetoacetyl-CoA reductase enzyme resulting from the expression of phaB leads to conversion of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA. Finally, pha synthase leads to synthesis of PHB from (R)-3-hydroxybutyryl-CoA ___!!source!!____.
In order to utilize acetic acid present in fermented human feces ___!!source!!____, we decided to incorporate the phaCAB operon. However, literature has shown that the rearrangement of operon to phaCBA leads to higher amount of production of PHB ___!!source!!____. Thus, we obtained the operon from <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1149051">BBa_K1149051</a> and rearranged the construct from phaCAB to phaCBA and added histidine tags. The iGEM suffix is at the end of the gene construct.
E. coli (BL21) as chassis ___!!source!!____
The part was inserted into pET29b vector downstream a T7 promoter and lac operon. Thus, the expression of the operon was induced using Isopropyl β-D-1-thiogalactopyranoside (IPTG). E. coli (BL21) was chosen as the chassis because of its ability in better protein expression. After transforming the bacteria with plasmid, colonies were selected and screened for presence of plasmid. A successful colony was then selected for experiments.
PHB from fermented "syn poo" supernatant ___!!source!!____
HPLC of PHB
Nile red suspension for confirmation of PHB
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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