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Team:Washington/Alkanes/Future/LuxCDE
From 2011.igem.org
Contents |
Alternative Routes for Fatty Aldehyde Production
Background
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 (Still working on this)
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)
