Difference between revisions of "Part:BBa K590064"

Revision as of 17:03, 26 October 2011

The FabBrick: FabH2, an enzyme for changing up fatty acid biosynthesis

This part encodes FabH2. [http://2011.igem.org/Team:Washington 2011 University of Washington iGEM Team] has produced even chain length alkanes using this part and the Petrobrick. In addition, expression of this part and the Petrobrick should theoretically produce branched chain alkanes, but we have not been able to demonstrate this effect, possibly due to the absence of the appropriate substrates in E. coli

Usage and Biology

Our previous attempts at using FabH2 utilized a construct where FabH2 was coexpressed with Aldehyde Decarbonylase and Acyl-ACP Reductase. This construct(BBa_K590030 did not result in even chain length alkane production, likely due to FabH2 toxicity, leading to low alkane yield( approximetly 10 mg/L vs. 170 mg/L for the Petrobrick).

In order to reduce these toxic effects, we cloned FabH2 onto a low copy number PSB3k3IPTG inducible expression vector to form the FabBrick. This construct was co-transformed with the Petrobrick in XL1-Blue E. coli. When FabH2 was induced by adding 5uM IPTG, a peak corresponding to the C16 were observed. This was confirmed from the MS spectrum, which had an overall fingerprint consistent with alkane, and a parent ion at a mass of 226, confirming the identity as C16 alkane. In addition, a peak corresponding to the C14 alkane was observed, completing the alkane spectrum from C13 to C17. This is the first tim e that even chain length alkanes have been recombinately produced. This part requires further optimization in order to further increase total alkane yield( currently at approximently 40 mg/L vs 170 mg/L for thePetrobrick), and to increase the amount of C14/C16 alkane yield, as current yield is only approximately 4 mg/L.

GCMS trace confirming C16 alkane produced only upon FabBrick induction.

MS spectrum verifies peak contains C16 alkane.

Sequence and Features