Difference between revisions of "Part:BBa K2260000"

Revision as of 03:11, 30 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 acetyl-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 BBa_K1149051 and rearranged the construct from phaCAB to phaCBA and added histidine tags. The iGEM suffix is at the end of the gene construct.

PHB Weights from fermented "syn poo" supernatant ___!!source!!____

HPLC of PHB

PHB produced from our construct was verified using crotonic acid digest __!!link to protocol!!__. The HPLC results verified that the white powder obtained from extraction of PHB from bacteria was indeed PHB. The following table shows the HPLC results:

High	Low	Arbitrary
High	Low	Arbitrary

Nile red suspension for confirmation of PHB