Can CRISPR Improve the Protein Secretion of Pichia pastoris
Poster Number
13A
Format
Poster Presentation
Faculty Mentor Name
Geoff Lin-Cereghino
Faculty Mentor Department
Biological Sciences
Abstract/Artist Statement
Pichia pastoris is a methylotrophic yeast that is widely used for protein expression, such as vaccines, anti-cancer proteins, and rheumatoid arthritis treatment proteins. Previously, the lab isolated a mutant strain called bgs13. It was assumed that the strain was a knockout; however, it was not. In this mutant, the bgs13 gene sequence was changed, and a modified protein was still being made that had kinase activity. The mutant had a lower than normal kinase activity and was a super secretor, meaning it was able to secrete increased levels of beta-galactosidase and other proteins.
Our goal is to make a true knockout of bgs13 and observe its effect on the secretion levels of reporter proteins. We have attempted to abolish kinase activity by making a knockout with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which targets the bgs13 gene in our studies. Having performed CRISPR, we are using colony PCR and sequence analysis to confirm that we have a strain in which the bgs13 sequence is edited to create an actual knockout. If we cannot isolate such a knockout strain, this would suggest that the bgs13 gene is necessary for survival. Thus, by searching for true knockouts of bgs13 with these methods, our results will help us figure out whether the bgs13 gene is essential for survival or can be knocked out to provide high levels of secretion.
Location
DeRosa University Center Ballroom
Start Date
27-4-2018 10:00 AM
End Date
27-4-2018 12:00 PM
Can CRISPR Improve the Protein Secretion of Pichia pastoris
DeRosa University Center Ballroom
Pichia pastoris is a methylotrophic yeast that is widely used for protein expression, such as vaccines, anti-cancer proteins, and rheumatoid arthritis treatment proteins. Previously, the lab isolated a mutant strain called bgs13. It was assumed that the strain was a knockout; however, it was not. In this mutant, the bgs13 gene sequence was changed, and a modified protein was still being made that had kinase activity. The mutant had a lower than normal kinase activity and was a super secretor, meaning it was able to secrete increased levels of beta-galactosidase and other proteins.
Our goal is to make a true knockout of bgs13 and observe its effect on the secretion levels of reporter proteins. We have attempted to abolish kinase activity by making a knockout with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which targets the bgs13 gene in our studies. Having performed CRISPR, we are using colony PCR and sequence analysis to confirm that we have a strain in which the bgs13 sequence is edited to create an actual knockout. If we cannot isolate such a knockout strain, this would suggest that the bgs13 gene is necessary for survival. Thus, by searching for true knockouts of bgs13 with these methods, our results will help us figure out whether the bgs13 gene is essential for survival or can be knocked out to provide high levels of secretion.