From 2011.igem.org

Revision as of 23:10, 15 September 2011

Alternative Routes for Fatty Aldehyde Production

Background

LuxCDE is an enzyme complex that produces a tetradecanal (14 carbon aldehyde) substrate by drawing from the fatty acid biosynthesis pathway. This tetradecanal substrate is to be catalyzed by the luxAB luciferase complex to produce light. The waste from the lucerferase is then sent back to luxCDE to be recycled. Since the aldehyde decarbonalse (ADC) uses aldehyde substrate to make alkane, we plan to use the luxCDE complex and ADC to synthesize alkane. The entire lux operon was submitted to the registry last year by Cambridge 2010.

Methods

PCR and Standard Biobrick Assembly

This method revolved around designing oligos to PCR amplify LuxCD and LuxE off of the LuxBrick; LuxE was not directly adjacent to LuxCD in the Luxbrick, and therefore had to be obtained separately. Sticky ends on the oligos would then allow us to obtain a LuxCDE using stitching PCR. We would then digest and cut the resulting fragment, ligate it into a similarly-cut vector, and use transformation to obtain the desired plasmid with LuxCDE embedded into it.

Gibson Assembly

We would design oligos to amplify C, D, and E off of the LuxBrick. Unlike the previous method, the oligos would have sticky ends of [ ] base pairs to prevent misannealing, and the fragment containing C would also contain the vector for ligation. A 3-piece Gibson assembly was then done on C, D, and E, and the product was then transformed into cells.

Gene Synthesis

Instead of using the LuxBrick to obtain C, D, and E, we would re-optimize the codons on C, D, and E to be more cloning friendly, then assemble them from oligonucleotides. PCR was then used to amplify the respective pieces, and each of the three pieces was then ligated into psb1C3. The end result is each of the three genes separated out and with codons that have a normal GC content.

Results

The PCR and Gibson methods failed at multiple steps for uncertain reasons. Since we do not have our LuxCDE construct, no method or assay for testing the hypothetical construct was developed as of this time. We hypothesize that LuxCD's abnormally high AT concentration may have caused misannealing for primers, causing primer-dimer errors and other problems, but no concrete evidence exists for this as of yet. For gene assembly, we have lowered the AT concentration of C, D, and E to more optimal levels for PCR.

[Still working on this, will be expanded once all experiments are complete]

Current Status

Attempts to clone LuxCDE using the PCR and Gibson method have been abandoned. Gene assembly was successful and each of the three luxCDE genes have been sequenced, biobricked and submitted.

Parts Submitted

LuxC, LuxD, LuxE

Image(s)

Subsection will be deleted and intergrated into article once images are found.