Date of Award

5-2017

Degree Type

Honors College Thesis

Department

Chemistry and Biochemistry

First Advisor

Sabine Heinhorst, Ph.D.

Advisor Department

Chemistry and Biochemistry

Abstract

In this project, a DNA construct was designed and developed to remove the first fifty amino acids of the CsoSCA protein in the chemolithotrophic bacterium Halothiobacillus neapolitanus. The csoS3 gene codes for a carbonic anhydrase enzyme (CsoSCA) that is unique to a structure called a carboxysome. Carboxysomes are polyhedral microcompartments where carbon fixation is housed. The carbonic anhydrase is a shell-associated protein that improves the catalytic efficiency of ribulose-bisphosphate carboxylase/oxygenase (RuBisCO), the enzyme that catalyzes the fixation of carbon. By deleting the first fifty amino acids of the carbonic anhydrase, the necessity of the amino acids in carboxysome function was evaluated.

The construct was designed by removing 150 bases from the coding sequence and by adding a kanamycin resistance cassette for selection of recombinant colonies. Regions of homology to the csoS2 and csoS3 coding regions were included for homologous recombination in E. coli DY330. Once the recombination event was successful, the isolated DNA was used for gene replacement in H. neapolitanus. Growth curves were generated for H. neapolitanus wildtype, a mutant in which csoS3 was deleted entirely, and the mutant generated with the designed construct (truncated csoS3). Comparing the growth curves of the wildtype and the mutants, it was found that while the mutant carrying the truncated carbonic anhydrase gene did not grow in air as well as wildtype, it grew considerably better than the deletion mutant. Deleting the codons for the first fifty amino acids of csoS3 does affect carboxysome function, but not as much as complete csoS3 deletion.

Included in

Biochemistry Commons

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