Team:UChicago/Human Practices
Human Practice
To understand the potential social concerns of the Pichia Project, we met with Dr. Thomas Purkarthofer from VTU Technology. VTU works with Pichia and produces several different high performance expression strains. Their work is concentrated in the field of Pichia Pastoris protein expression. Dr. Purkarthofer brought up many points which not only informed our research, but guided our experiments. The primary purpose of our project is to create a plasmid which can attach to the centromeres, thus ensuring that it is distributed to all daughter cells. In order for our plasmid to be useful however, Dr. Purkarthofer advised us to create experiments that allow us to verify that the entirety of the plasmid is integrated into the chromosome. For example, we considered his suggestion to linearize the plasmid before transformation so the DNA is incorporated via homologous recombination. Additionally, we designed sectoring assays to observe which yeast colonies lost the plasmid. Our destination sequence homology on the plasmid will be directed towards the centromere. VTU uses genomic integration to integrate DNA into a desired locus. While this method is not guarantee, Dr. Purkarthofer said that they’d been able to get consistent success. One of the concerns Dr. Purkarthofer had was concerning the usefulness of the plasmid. We considered his points and examined how our construct might be more directly applicable. One way in which we can do this is by measure mRNA levels to observe of its level of transcription. We could also measure the amount of protein produced in a volume of culture. By examining the plasmid’s transcription, we would gain valuable knowledge about each specific clone. However, Dr. Purkarthofer cautioned that high mRNA levels don’t always translate into high protein production levels. We used Dr. Purkarthofer’s points to guide our focus going forward. We’d like to compare our construct with commercially available ones already designed for genomic integration. This would allow us to fully examine its practicality. Furthermore, it would be greatly beneficial if we could provide a construct with increased transformation efficiency. By meeting with Dr. Purkarthofer from VTU we were able to guide our experiments and set goals for the future that ultimately will result in a more practical centromeric plasmid.