To reduce nitrates from the environment, we wanted the most efficient driver of nitrate dependent reactions we could find. This involved a few key features: first, the protein itself that would become our nitrate sink, then our promoter. The model essentially functions like this—if there is freestanding nitrate at an extreme excess in the environment, then within the bacteria, there must be a sink that collects the nitrate in some form that then becomes trapped and unable to return to the environment as free standing nitrate. This sink’s action will be driven forward by the promoter, while the promoter encourages the production of the protein next to it, the sink will be active. Eventually, the cell’s internal supply of nitrogen will run out, and to satisfy the demand created by the sink, the cell will intake the free nitrates in the environment.
Our constructs were very large. When we first ordered them, we were made aware that though the constructs technically fit the parameters given by IDT, they were so bulky that they couldn’t make the full amount to give us. This didn’t technically cause any issues outright, but the constructs being big and unwieldy became a theme throughout the rest of the wetlab work. cphA was so large that it was ordered in two pieces. cphB was marginally smaller. When we attempted to insert cphA1 and cphA2 into our plasmids and then transform into bacteria, we realized that only our cphB had been a success. However, since there was one small colony that seemed to be viable, we sampled from there and attempted to grow up our supply of cphA. Eventually, once we managed to combine cphA with cphB and transform into bacteria, we got our DNA sequenced. We then realized that our DNA was not what we expected. We witnessed a twenty base pair deletion in the middle of the sequence. We attempted to ensure that this was not an issue with our cloning; we redid the Gibson Assembly, retransformed, and witnessed the same issue over and over again. At this point, we assumed that something was wrong with the constructs we were given; we were, after all, informed from the beginning that for whatever reason, this construct seemed to stress IDT’s capabilities. To fix the issue, we used the Q5 Site Directed Mutagenesis Kit in order to repair our construct. We had to order corresponding primers to work with the kit. We performed the protocol once, transformed, and sequenced, then we realized that the first attempt wasn’t successful. However, once it was attempted a second time, the twenty base pair deletion had been fixed, determined through sequencing. We proceeded with Golden Gate, then encountered an overgrowth after transformation, which we suspected was due to old plates losing their antibiotic potency.