Difference between revisions of "Part:BBa K590064"

Latest revision as of 00:44, 2 November 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 (approximately 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 pSB3K3 IPTG 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 time that even chain length alkanes have been recombinantly produced. This part requires further optimization in order to further increase total alkane yield (currently at approximately 40 mg/L vs 170 mg/L for the PetroBrick), and to increase the amount of C14/C16 alkane yield, as current C14/C16 yield is only approximately 4 mg/L.

GCMS trace confirming C16 alkane produced only upon FabBrick induction. Black trace from the PetroBrick alone. Red trace from the PetroBrick + uninduced FabBrick. Green trace from the PetroBrick + induced FabBrick.

GCMS trace confirming C14 alkane produced only upon FabBrick induction. Black trace from the PetroBrick alone. Red trace from the PetroBrick + uninduced FabBrick. Green trace from the PetroBrick + induced FabBrick.

Reference

Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 126
Illegal AgeI site found at 1954
Illegal AgeI site found at 2593
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 1999

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-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 [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590025 Petrobrick].  In addition, expression of this part and the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590025 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''
+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 [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590025 PetroBrick].  In addition, expression of this part and the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590025 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 the[https://parts.igem.org/wiki/index.php?title=Part:BBa_K590025 Petrobrick]. Expression of FabH2 on the same  high copy number constiuitive plasmid as the PetroBrick( as in Part [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590030 BBa_K590030]) results in slow cell growth( insert picture), and low alkane yield( under 10 mg/L vs. approximately 170 mg/L in the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590025 Petrobrick]).
+Our previous attempts at using FabH2 utilized a construct where FabH2 was coexpressed with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase]. This construct([https://parts.igem.org/wiki/index.php?title=Part:BBa_K590030 BBa_K590030]) did not result in even chain length alkane production, likely due to  FabH2 toxicity, leading to  low alkane yield (approximately 10 mg/L vs. 170 mg/L for the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590025 PetroBrick]).
-In order to reduce these toxic effects, we cloned FabH2 onto a low copy number PSB1C3 [https://parts.igem.org/wiki/index.php?title=Part:BBa_K314103 IPTG inducible PetroBrick ]. This construct was co-transformed with  the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590025 Petrobrick] in XL1-Blue ''E. coli''.  cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in [http://2011.igem.org/Team:Washington/alkanebiosynthesis#100mL_M9_minGlucose_Media_Prep  M9 production media] + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/Petrobrick cultures, and on cells expressing only the PetroBrick.
+In order to reduce these toxic effects, we cloned FabH2 onto a low copy number pSB3K3 [https://parts.igem.org/wiki/index.php?title=Part:BBa_K314103 IPTG inducible expression vector] to form the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590064 FabBrick]. This construct was co-transformed with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590025  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 time that even chain length alkanes have been recombinantly produced. This part requires further optimization in order to further increase total alkane yield (currently at approximately 40 mg/L vs  170 mg/L for the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590025 PetroBrick]), and to increase the amount of C14/C16 alkane yield, as current C14/C16 yield is only approximately 4 mg/L.
-[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS  trace confirming C16 alkane produced only upon FabBrick induction. ]]
-There was no significant difference between the GC peaks in the PetroBrick extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When FabH2 is induced, we see a strong new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinately, and expands the PetroBrick system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 1mg/L), and future optimization is needed to increase even chain length production levels.
+[[Image:FabBrickGCMS.png|left|440px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction.  Black trace from the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590025 PetroBrick]  alone. Red trace from the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590025 PetroBrick] +  uninduced FabBrick. Green trace from the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590025 PetroBrick] + induced FabBrick.]]
-[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]
+[[Image:Washington_2011_C14spectrum.png|right|440px|thumb|GCMS trace confirming C14 alkane produced only upon FabBrick induction. Black trace from the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590025 PetroBrick] alone. Red trace from the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590025 PetroBrick] + uninduced FabBrick. Green trace from the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590025 PetroBrick] + induced FabBrick.]]
+<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
+==Reference==
+Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. Choi KH, Heath RJ, Rock CO. J Bacteriol. 2000 Jan;182(2):365-70.