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

Acuff, Jennifer

Committee Member/Second Reader

Pinto, Ines

Committee Member/Third Reader

Kumar, T.K.S.

Abstract

Methanogens are nitrogen fixing and methane producing archaea that play a key role in the cycling of carbon and nitrogen on earth. The global carbon and nitrogen cycle is vital to all life which makes the methanogen a primary organism of study. Methanogens are the only archaea capable of fixing nitrogen, and in order to do so, methanogens use a vital enzyme called the nitrogenase which fixes dinitrogen (N2) to ammonia (NH3). Expanding our understanding on metabolic processes of methanogens allows us to understand their environmental impact and advance their use in biotechnological applications. Methanosarcina acetivorans is the model used to study nitrogenase and nitrogen fixation in methanogens. In order to analyze factors affecting diazotrophic growth, and new strain of M. acetivorans was developed. DJL74 is a knockdown strain that was created using the CRISPRi-dCas9 system, where dCas9 is blocking the expression of nitrogenase to eliminate nitrogen fixation. Previous growth studies with the DJL74 strain were performed to test a variety of amino acids on their ability to be utilized as a fixed nitrogen source; however, it was found that cells that were given cysteine were unable to grow in conditions where ammonium chloride was absent. To further investigate the abnormal lack of growth, experiments were conducted where varying concentrations of cysteine were provided to tubes of inoculated DJL74, with ammonium chloride and no cysteine as the positive growth control and neither cysteine nor ammonium chloride as the negative control. Growth was monitored over a period of approximately two weeks using optical density recordings. Results showed that there may be threshold for an inhibitory effect at around 5mM of cysteine, with varying growth below 5mM and no growth with concentrations greater than 5mM. When ammonium chloride is present, normal growth is permitted; however, without the nitrogen source present, there is no growth diazotrophically. In order to determine the cause of these effects, it will be useful to determine whether levels of cysteine is preventing cells from overcoming dCas9 suppression or if cysteine has an inhibiting effect on the metabolic process of nitrogen fixation.

Keywords

methanogen; cysteine; nitrogen fixation; nitrogenase

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