Date of Award
Spring 5-2017
Degree Type
Honors College Thesis
Department
Chemistry and Biochemistry
First Advisor
Sabine Heinhorst
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.
Copyright
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Recommended Citation
Dillistone, Dana L., "Altering the Structure of Carboxysomal Carbonic Anhydrase CsoSCA to Determine the Necessity of the N-terminal Domain in CsoSCA Function" (2017). Honors Theses. 520.
https://aquila.usm.edu/honors_theses/520