Discipline: Biological Sciences
Subcategory: Biomedical Engineering
Heather Waters - University of Wisconsin – Washington County
Co-Author(s): Leah Thomas, Shu Pan, and Jennifer Reed
Isocitrate dehydrogenase (icd) catalyzes an important step in the citric acid cycle, which generates energy (in the form of ATP) and chemicals needed for cellular growth. In this project, we wanted to improve the enzyme activity of icd by protein engineering. The icd gene is essential for growth on glucose minimal media, and so Escherichia coli mutants lacking icd are unable to grow in this condition. The first part of my project was to create plasmids with low expression levels of icd, which could be expressed in a mutant E. coli strain which lacks icd (delta icd). The delta icd mutant was generated and confirmed to be unable to grow on glucose minimal media. We then generated a variety of icd expression plasmids using PCR, Gibson cloning, and transformation. The plasmids were extracted and transferred into the delta icd mutant. Since icd is essential for growth, the plasmids with the lowest expression should have the slowest growth rates. The mutants containing icd plasmids were grown in a plate reader for 24 hours and the corresponding growth rates were calculated. The second part of the project was to generate random variants of the icd gene, which was accomplished using error prone PCR. Future work, will be to test growth of delta icd mutants harboring plasmids with different icd sequence variants and measuring growth. Variants of icd that improve growth will be sequenced and their catalytic properties measured.
Funder Acknowledgement(s): NSF 1053712 and NSF EFRI1240268
Faculty Advisor: Jennie Reed,