Date of Graduation

5-2018

Document Type

Thesis

Degree Name

Bachelor of Science in Biological Engineering

Degree Level

Undergraduate

Department

Biological and Agricultural Engineering

Advisor/Mentor

Haggard, Brian E

Committee Member/Reader

Lessner, Dan

Committee Member/Second Reader

Kim, Jin-Woo

Abstract

Methanogenesis is the biological production of methane. Only anaerobic archaea known as methanogens are capable of such a metabolic feat. They have strict living conditions and substrate sources which determine their rate of metabolism. This is of particular importance from a greenhouse gas reduction perspective or biogas capturing perspective. One of the best ways to optimize methanogen methane production is via genetic manipulation. The current procedures are timely though, therefore a faster cloning processes should be developed. The objective of this study was to optimize a premade genetic transformation kit known as the Gibson Kit. The Gibson Kit was supposed to ease the work of genetic manipulation by combining several linear chunks of DNA together at once via the use of sequence overhangs. The resulting plasmid from the Gibson could then be transformed into E. coli where E. coli is supposed to replicate the plasmids extremely quickly. Afterwards, that plasmid could then transform methanogens. Several baseline PCR (polymerase chain reaction) transformations were performed to linearize and amplify the desired plasmid of pNB72.3. PCR amplifications of the desired gene segments which would be added to the plasmid were also performed. The desired gene segments being assembled were supposed to take out the production of Cytochrome C within the methanogen electron transport system by deleting the ccmf gene. Several PCR experiments were carried through without success. The cause of the failures included the primers “hair pinning” at the recommended annealing temperatures, primers being too nonspecific, and primers unable to perform efficiently at the recommend annealing temperatures. After several tries of no success, the Gibson Kit was tested without PCR linearization of the circular plasmid. Instead, standard digestion was relied on. With standard electro competent cell transformation and antibiotic screening, the Gibson product was then tested for successful transmission of the plasmid to the E. coli. Results were negative; therefore, the optimization of faster cloning techniques was unsuccessful, but it will help guide future efforts.

Keywords

methanogens; Gibson Kit; PCR; genetic cloning; methane production; cytochrome C.

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