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The Microbiology
Group of the Environmental Research Division applies
Biochemistry and Microbiology
to the production of proteins and chemicals.
| | Tools for the Production of Proteins
Part of the research of the Microbiolgy Group
focuses on developing new expression vectors and culture methods for the production of proteins for
structure determination as part of an NIH-funded in structural biology center,
the Midwest Center for Structural Genomics.
The expression vectors, designed to fit the requirements of robotic cloning and purification methods, drive the production of proteins
in bacterial cells to generate sufficient amounts of material to purify.
New culture methods using common 2-L plastic soda bottles as disposable culture vessels greatly reduce the time
and effort (and cost) needed to produce the proteins.
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Labor- and cost-saving approaches for growing cells in 2-L soda bottles
 
Use of the bottles cuts the cost of producting proteins
in half, and gives staff more time for purifying and crystallizing
proteins. Because two antibiotics are present in
the medium, and a large inoculum of health cells
are introduced, sterilization is not necessary. After growth, the bottles
are simply discarded. Publications...
Expression vectors tailored for robotic cloning
and automated purification
A series of ligation-independent cloning (LIC) vectors, the pMCSG vectors,
was developed for high-throughput production of proteins based on criteria defined by the demands of purification,
crystallization and structure determination. The base vector, pMCSG7, shown here, encodes an N-terminal his-tag followed
by a spacer, the TEV protease recognition sequence, and a LIC site based on the enzyme SspI. Additional restriction sites in and
around this region allow insertion of other proteins or peptides into the leader sequence or transfer of the entire region to
different vector backgrounds. Publications...
Production of Chemicals from Renewable Feedstocks
Additional activities of the Microbiology Group have improved potential new routes to
industrial chemicals from renewable, biological feedstocks such as agricultural sugars or fats.
Genetic engineering and enzymology developed new strains or enzymes with applications in the production
of succinic acid and vitamin C from glucose, long-chain dicarboxylic acids from fatty acids, and lactic acid from
mixed carbohydrates of lignocellulosic hydrolysates. Where appropriate, engineering was guided by the requirements
of downstream separations processes. The research generated several patents, and in the case of succinic acid production, received an R&D 100 award.
Funding from the U.S. Department of Energy's Office of Industrial Technology and the U.S. Department of Commerce's Advanced
Technology Program supported these projects. Patents and Publications...
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Mutation of three genes of E. coli converts wild type fermentation (left) to succinic acid production (right)
Mutation of ldhA, pflB, and ptsG converted the typical mixed-acid fermentation of E. coli, which produces
predominantly formate, acetate and ethanol, to a novel distribution of fermentation products in which half of the glucose was converted to succinate.
Patents and Publications...
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