Title

Can CRISPR Improve the Protein Secretion of Pichia pastoris

Poster Number

13A

Lead Author Major

Pre-Dentistry

Lead Author Status

Sophomore

Second Author Major

Pre-Dentistry

Second Author Status

Sophomore

Format

Poster Presentation

Faculty Mentor Name

Geoff Lin-Cereghino

Faculty Mentor Email

glincere@pacific.edu

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

This document is currently not available here.

Share

COinS
 
Apr 27th, 10:00 AM Apr 27th, 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.