Difference between revisions of "Part:BBa K2260000"

Revision as of 06:10, 27 October 2017

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!!____. 

This part contains the naturally occurring phaC, phaA, and phaB operon rearranged to phaC, phaB, and phaA. It also contains his tags and ribosome binding sites (RBS). The iGEM suffix is at the end of the gene construct. Transcription along the operon leads to expression of the enzymes: 3-ketothiolase, acetoacetyl-CoA reductase, and pha synthase. These enzymes are involved in the pathway that converts acetyl-CoA to polyhydroxybutyrate (PHB). The operon was rearranged based on results from literature <source>, which showed that the phaCBA operon leads to higher production of PHB compared to phaCAB.

This part can be used to utilize the end-product of glycolysis pathway i.e. acetyl-CoA. After a series of three reactions, PHB is obtained.

This part was used in our project to utilize acetic acid, which is present in the supernatant collected from fermented synthetic feces. The part was tested using E. coli (BL21) as the chassis. The bacteria containing the vector was The amount of PHB produced from E. coli at known cell density was recorded using mass balance. Furthermore, HPLC and nile red staining was used to confirm the presence of PHB.

Sequence and Features