As our institute remains closed to students due to the coronavirus pandemic, we would not be able to perform the experiments we have planned within time for iGEM 2020. This section is a discussion about what results we might expect from our experiments and our contingency plans in case some of them don’t go as planned.
We had initially performed some preliminary experiments in March to verify the function of wild-type pyocins against susceptible P aeruginosa strains and their effect(or lack thereof) on A. baumannii. However, evacuation of the campus was ordered shortly after planning of this. The experiment ended up failing, as we tried to rush through them. Inducing expression of pyocins in PAO1 is done by addition of mitomycin C, a mutagen that triggers the SOS pathway. The order for the reagent did not arrive in time, and so we tried to induce expression through oxidative stressor methylviologen. Pelleting the proteins from the PAO1 lysates showed that there was no expression induced.
Gibson Assembly being a highly reliable process should not pose any problems in terms of results. We chose to use the two-step Gibson protocol, which has been shown to reliably assemble upto 15 fragments. However, since this is our first time doing Gibson, we contemplate the possibility of a learning curve and optimization.
In the unlikely case that cloning the synthetic DNA fails, we would circumnavigate this by cloning the native genes from P. aeruginosa. PCR cloning of the pyocin gene cluster from PAO1 followed by replacement of the tail fiber gene with a fusion tail fiber gene by restriction-ligation would be done. The gene products would be identical to those from the synthetic DNA.
However, the native gene cluster contains many illegal restriction sites which would have to be edited through site-directed mutagenesis. This is a rather lengthy process which we hope to avoid, and hence our primary plan is the synthesis method.
In the case that the produced Seekercins do not show positive results on any of the A. baumannii isolates, we would proceed by producing pyocins with alternate fusion tail fibers made from other Acinetobacter-specific phages. A new A. baumannii phage is characterized in the literature every few months, and selecting new candidates may be possible. Another approach will be to try other fusion sequences of the AP22 tail fiber .We would use different lengths of N-terminal pyocin tail fiber and C-terminal AP22 tail fiber in combination and perform the spot test to determine which of those would be viable.