Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.
To be able to use Gibbson Assembly we needed to add overlapping overhangs to our construct. The first time we designed them, we chose XbaI as restriction enzyme and primers that would match our constructs and the restriction site. We then faced the problem, that the G-C content was too high to ensure good PCR results. To overcome this issue we designed a two-step PCR for the primers for acetate, butyrate and prpR: In the first step we used primers that added sequences that are low in G-C to the primers for our construct and therefore enabled another set of primers in a secondary PCR to then bind to those sequences. After trying different temperatures and primer concentrations, we still had troubles doing the first step PCR, indicating that the G-C content was already too high for the first set of primers to bind. We solved the problem by choosing another restriction site based on a low G-C content at the neighbouring bases and used the restriction enzyme Hind3. Because of the change we could directly design primer that added the needed overhangs in one step.
Since bacteria also produce endogenous acetate, we were afraid that this would influence our measurements of external acetate. One idea we had, was to produce an acetate knockout. We discussed our ideas with Prof. Winkler, who told us that we could use the CRISPR-Cas9 system for that, but that we would need to be careful to ensure those modified bacteria would still need to be able to live without the acetate. We then thought about adding acetate to the media and remove it before starting the tests. In the future we would then need to measure, how long those bacteria can survive without endogenous acetate and when adding it to the media, how we would need to deprive our bacteria before starting the test series.