Dilorio, Alexander A.
Escherichia coli strain BL12 was obtained from Dr. Carlos B. Hirschberg at Boston University. BL 12 constitutively expresses the human GDP-fucose-transporter, a Golgi membrane protein. By definition, a constitutively expressed protein is produced at a constant rate regardless of any physiological condition or concentration of substrate. The goal of this project was to assess the production rate of the GDP-fucose transporter by varying only the growth rate of strain BL 12 using a controlled limiting feed of carbon source, in this case, glucose. For a true constitutive protein, the production rate of protein should be proportional to the growth rate. Whereas, for a typical induced recombinant protein, the growth rate is inversely proportion to the production rate of the recombinant protein. Three different growth rates were tested based on growth studies preformed in shake flasks. Growth rates corresponding to a doubling time of 2, 2.5 and 3 hours were tested. The maximum doubling time of the strain in the completely defined medium used was approximately 2 hours. A growth rate of 0.347 hr? corresponding to a 2 hour doubling time, resulted in the highest biomass generation and a final OD600nm of 93, suggesting that a cell which produces a constitutive protein can be manipulated to increase yield by varying the growth rate.
Worcester Polytechnic Institute
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Biology and Biotechnology