Date of Graduation

5-2022

Document Type

Thesis

Degree Name

Bachelor of Science

Degree Level

Undergraduate

Department

Biological Sciences

Advisor/Mentor

Lessner, Daniel

Committee Member/Reader

Willson, John D.

Committee Member/Second Reader

Millett, Francis

Committee Member/Third Reader

Clay, Matthew

Abstract

As of 2016, the genomes of over sixty species of methanogenic archaea have been sequenced.4 Research on the genomes of methanogens such as M. acetivorans led to the characterization of numerous genes with unknown functions. Genes similar to those of nitrogenase have been shown to possess functions unrelated to nitrogen fixation, such as F430 biosynthesis genes. 5,6,7 My research focused on the uncharacterized gene clusters ma2032-33 and ma1631-33 (Figure 1). These genes have conserved nitrogenase domains: ma2032 has fused nifH and nifD domains, while ma2033 has a nifD domain. The ma1631-33 gene cluster contains two nitrogenase homologs and one iron protein homolog. I hypothesized the ma2032-33 and ma1631-33 clusters are not involved in the process of nitrogen fixation in M. acetivorans because of the numerous nitrogenase-like genes that have unrelated functions. To test this hypothesis, I used the recently developed CRISPRi-dCas9 system to generate M. acetivorans strains in capable of expressing either ma1631-33 or ma2032-33.

Keywords

nitrogenase; methanosarcina acetivorans; nitrogen fixation; CRISPRi-dCas9

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