Unfortunately, we ran out of time before we could sufficiently characterize or build a number of our parts. However, this was still a learning experience that provided us with a lot of information
We were able to successfully biobrick and submit the parts here
We sucessfully showed expression of the gut peptides: GLP-1, PYY, and Ghrelin.
In order to quantify the secretion of our cells, we put his-tags to the end of the constructs in order to perform a Western Blot. In order to compare how much protein was being secreted and how much remained inside the cell, we spun the cells down and did blots for the cell pellet and supernatant. However, all of our attempt to measure protein levels in the supernatant came back negative. We suspected this was due to low concentration rather than no secretion as the signal peptides have been shown to work for others.
However, we were able to show that the gut-peptides themselves are being expressed as the dot blots for these came back positive. As such, future goals for the project would involve better ways to measure secretion levels in the environment of the cells. We did start the design of an experiment to quantify secretion activity by putting luciferase downstream of the signal peptide and using fluorescence as a proxy for activity, but the time frame did not allow us to do so.
Sequencing and PCR
Much of the plasmids that we sent out for sequencing came back with either poor quality scores or a sequence that did not align properly with the expected sequence. As such, it seems that there seems to be something going wrong when we are amplifying genes out, especially when primers are designed to add flanks. In addition, the primers seemed to have low specificty with G-blocks and resulted in very poor quality PCR. Thus, for the future, primers with higher specifity will have to be used to improve yields
The efficiencies of the electroporations we were performing may have also been an issue, but we have not successfully trouble-shooted the issue here