Difference between revisions of "Part:BBa K590064"

Revision as of 16:44, 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

FabH2 is from Bacillus subtilis. The FabH family of proteins initiates fatty acid elongation by converting an Acyl-CoA into an Acyl-ACP, with is extended by 2 carbon units to form longer chain length fatty acids. Normally, FabH proteins use a simple 2-carbon acetyl-CoA to start fatty acid biosynthesis, resulting in linear fatty acids. However, FabH2 can also use Isobutyryl-CoA, Isovaleryl-CoA, and 2-Methylbutyryl-CoA (products from Valine, Leucine, and Isoleucine degradation), resulting in 2-methyl branched fatty acid production. In addition, FabH2 has been hypothesized to start fatty acid elongation with a straight 3-carbon unit(propionyl-CoA), yielding odd chain length fatty acids, which could be converted into even chain length alkanes by thePetrobrick. Expression of FabH2 on the same high copy number constiuitive plasmid as the PetroBrick( as in Part BBa_K590030) results in slow cell growth( insert picture), and low alkane yield( under 10 mg/L vs. approximately 170 mg/L in the Petrobrick).

In order to reduce these toxic effects, we cloned FabH2 onto a low copy number PSB3k3IPTG inducible PetroBrick . 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.

GCMS trace confirming C16 alkane produced only upon FabBrick induction.

MS spectrum verifies peak contains C16 alkane.

Sequence and Features