Publication Date

Fall 2014

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Biomedical, Chemical & Materials Engineering

Advisor

Melanie McNeil

Keywords

E. Coli, glucose, mlc, modulation, recombinant protein, therapeutic protein

Subject Areas

Molecular biology

Abstract

When Escherichia coli (E. coli) is grown in the presence of excess glucose, acetate is produced, oftentimes as an undesired by-product. Mlc is a global repressor for sugar transporters in E. coli, including glucose. This body of work examines the overexpression of Mlc via expression vectors in E. coli cultures under constitutive and inducible promoters. Sequence changes to the translational start codon and codon 52 of the mlc sequence inserted in the expression vectors were introduced. These changes were evaluated for their impact on glucose uptake rates, acetate production, and overall cell growth when Mlc was overexpressed in E. coli cultures in the presence of excess glucose. Furthermore, expression vectors carrying the mlc gene versions were co-transformed with a plasmid encoding for a therapeutic protein in order to study the impact of Mlc overexpression on the production of the therapeutic protein. Results showed varied levels of Mlc overexpression; however a correlation was drawn between increased Mlc expression and decreased acetate production as a result of slower glucose uptake into the cell. This characteristic resulted in improved cell growth in the form of higher density cultures. In addition to growing to higher cell densities, a 1.7-fold increase of therapeutic protein production was observed in cultures overexpressing Mlc, compared to the control.

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