Cloning of the Genes for Colicin v Production and Immunity and Identification of the Gene Products
Frick, Kevin Kurt
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https://hdl.handle.net/2142/71159
Description
Title
Cloning of the Genes for Colicin v Production and Immunity and Identification of the Gene Products
Author(s)
Frick, Kevin Kurt
Issue Date
1983
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Abstract
The genes for colicin V production and immunity from pColVB188 have been cloned into the amplifiable vectors pMB9, pBR322, pMK16, and into the bacteriophage M13mp8. The genes are closely linked and can be isolated on a deoxyribonucleic acid fragment of about 900 base pairs. The use of the transposon Tn5 to mutagenize this cloned DNA sequence has led to the construction of a derivative plasmid which conferred immunity to colicin V but not the ability to produce this colicin. The gene products specified by these cloned genes were analysed by use of minicells, an in vitro coupled transcription-translation system, and in vivo in maxicells. Analysis of the gene products has led to the conclusion that the polypeptide associated with immunity has a molecular weight of about 6,500, whereas the colicin has a molecular weight of approximately 4,000. The small size of the active colicin has been confirmed by use of sucrose gradient sedimentation. Attempts were made to purify the colicin for characterization by use of molecular seive and ion exchange chromatography. The insertion of intact colicin V production and immunity genes into M13mp8 has put colicin production under partial control of the lacOP region. The application of base-specific chemical modification has permitted the region immediately adjacent to the Tn5 insertion to be sequenced.
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