PURCHASE OF FERMENTER AND OLIGONUCLEOTIDE SYNTHESIZER

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Funds are requested for the purchase of a 250 liter fermenter and an oligonucleotide synthesizer. The primary application for these instruments will be in site-specific oligonucleotide-directed mutagenesis and isolation of the mutant proteins in large quantities. The instruments will be multi-user and will be available generally to the scientific community in Houston when use will not interfere with the projects of the investigators listed. In addition to the production of mutant proteins in large quantities, the fermenter will be utilized in several other projects which require large-scale fermentation (production of enzymes from Clostridia, production of bacteria for purification of wild-type proteins for routine use, and growth of bacteria for large-scale plasmid purification). The oligonucleotide synthesizer is required for the production of large quantities of specified sequences of DNA's; these will be used primarily for site-specific mutagenesis by standard methods using coding or recognition regions cloned in plasmids or in various Ml 3 vectors. A second application is the generation of synthetic oligonucleotides of various composition for use in binding studies in a variety of systems under investigation in the laboratories of the participating researchers. The coupling of these two instruments is essential for the generation of large quantities of selected mutant proteins; production of the mutant strains with site-specific oligonucleotides and the capacity to grow large quantities of cells will provide the resources to isolate sufficient amounts of the proteins for detailed chemical and physical characterization. Production of mutants for binding and repressor proteins, calmodulin, and promoter sequences, as well as metabolic studies of Clostridia strains, will be undertaken with these instruments. These important projects are entirely dependent on the availability of one or both of these instruments.