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Team:Washington/Alkanes/Future/LuxCDE
From 2011.igem.org
Contents |
Alternative Routes for Fatty Aldehyde Production
Background
LuxCDE is an enzyme complex that produces a 14 carbon aldehyde from a 14-carbon Acyl-ACP. Standardized into BioBrick format by a previous iGem team (Cambridge, 2010), LuxCDE should be easily cloned, and may produce a better yield and specificity of alkanes than AAR.
Methods
- PCR
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 (mostly accurate, need to make sure on some stuff)
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 optimize C, D, and E, then assemble them ourselves. PCR was then used to amplify the respective pieces, and each of the three pieces was then digested and transformed into cells.
[Still working on this, will be expanded after experiments are complete]
Results
The PCR and Gibson methods failed at multiple steps for uncertain reasons. 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
Gene assembly is still a work in progress, sequencing-matching is being done at the time of writing.
