Date of Graduation

5-2020

Document Type

Thesis

Degree Name

Bachelor of Science

Degree Level

Undergraduate

Department

Biological Sciences

Advisor/Mentor

Lessner, Daniel

Committee Member/Reader

Robinson, Samantha

Committee Member/Second Reader

Kral, Timothy

Committee Member/Third Reader

Thallapuranam, Suresh

Abstract

Methanoperedens nitroreducens (MPEBLZ), an archaeal methanotroph and close relative of Methanosarcina acetivorans, contain numerous cytochrome c proteins. However, difficulty in using these organisms as a model for cytochrome cresearch has created a pressure to express cytochrome c proteins in an organism that is much easier to work with. A punitive cytochrome c protein (MPEBLZ_04274) from M. nitroreducens was attempted to be cloned into a model methanogen M. acetivorans as well as Escherichia coli. Cytochrome c proteins are important for many metabolic processes within anaerobic archaea. In order for a mature cytochrome c to be formed heme must be translocated out of the cell and covalently attached to thiol bonds within CXXCH binding motifs within the protein. Preliminary data suggests that thioredoxin system components, which catalyzes redox reactions within the cell, may be important for the insertion of heme in cytochrome c proteins. To investigate this, CRISPR-dCas9 targeted repression of trx3, trx6, and trx7 was carried out to elucidate the potential impact that these thioredoxins have on the maturation of cytochrome c proteins as well as the ability of the cell to overcome general oxidative stressors.

Keywords

Methanosarcina acetivorans; cytochrome c; thioredoxin; oxidative stress; CRISPR-cas9

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