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Publication Date

Fall 2011

Degree Type

Thesis - Campus Access Only

Degree Name

Master of Science (MS)

Department

Chemical and Materials Engineering

Advisor

Claire Komives

Keywords

Bacillus methanolicus, Electroporation, Green Fluorescent Protein (GFP), Intracellular pH, Recombinant protein expression

Subject Areas

Chemical engineering; Microbiology

Abstract

Microbial biocatalysts are used in a wide range of industries to produce large scale quantities of proteins, amino acids, and commodity chemicals. Most of these processes use glucose or other low-cost sugars as the substrate. Previous research with Bacillus methanolicus has shown sustained growth at 50°C using methanol as its carbon and energy source, which results in reduced feed and utility costs. In this study, recombinant protein production by B. methanolicus was explored. This was achieved by transforming wild-type B. methanolicus with plasmid pNW33N-mdh-GFPuv. In addition to confirmation of a successful transformation, results from agarose gel electrophoresis revealed that the native plasmid pBM19 of wild-type B. methanolicus was maintained in the transformed B. methanolicus along with plasmid pNW33N-mdh-GFPuv. Plasmid pBM19 has been known to include essential genes for growth of B. methanolicus on methanol. Using high performance liquid chromatography (HPLC), it was confirmed that the transformed B. methanolicus utilized methanol as its carbon and energy source.

Due to the effect of pH on green fluorescent protein (GFP) emission at elevated temperatures, the GFP expression in the transformed B. methanolicus was analyzed by lowering the incubation temperature from 50°C to 40°C for 30 minutes. By growing the transformed B. methanolicus at various initial methanol concentrations, it was determined that recombinant protein production was the highest at 200 mM methanol.

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