Development and application of a gene transfer system in Clostridium perfringens
Allen, Steven Paul
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Permalink
https://hdl.handle.net/2142/23624
Description
Title
Development and application of a gene transfer system in Clostridium perfringens
Author(s)
Allen, Steven Paul
Issue Date
1990
Doctoral Committee Chair(s)
Blaschek, Hans-Peter M.
Department of Study
Agriculture, Food Science and Technology
Biology, Microbiology
Discipline
Agriculture, Food Science and Technology
Biology, Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Agriculture, Food Science and Technology
Biology, Microbiology
Language
eng
Abstract
Initially, electroporation-induced transformation of intact cells of C. perfringens 3624A-Rif$\sp{\rm r}$/Str$\sp{\rm r}$ with plasmids pAM$\beta$1 and pHR106 resulted in transformation efficiencies of 1.4 $\times$ 10$\sp2$ and 1.2 $\times$ 10$\sp3$ transformants/$\mu$g DNA, respectively. An examination of parameters involved in the electrotransformation process determined that the following factors improved the transformation efficiency of C. perfringens 3624A-Rif$\sp{\rm r}$/Str$\sp{\rm r}$: (1) a reduction in cuvette volume (DNA and cell suspension) to 0.8 ml, (2) use of a 1 $\mu$g/ml concentration of transforming DNA, (3) use of late-logarithmic phase cells, (4) three-fold concentration of cell density (3.0 $\times$ 10$\sp8$ CFU/ml), and (5) a reduction in the pH of the expression and selective plating medium to 6.4. Application of the optimized conditions resulted in transformation efficiencies for C. perfringens 3624A-Rif$\sp{\rm r}$/Str$\sp{\rm r}$ ranging from 7.1 transformants/$\mu$g DNA for plasmid plP401 to 9.2 $\times$ 10$\sp4$ transformants/$\mu$g DNA for plasmid pAK201. The greatest transformation efficiency obtained using pAK201 was 9.8 $\times$ 10$\sp6$ transformants/$\mu$g DNA for C. perfringens strain 13. In addition to C. perfringens 3624A-Rif$\sp{\rm r}$/Str$\sp{\rm r}$, eight out of eleven C. perfringens strains examined were transformable using the optimized protocol. Examination of nuclease activity suggested that DNase does not appear to be a factor in the C. perfringens strain-specific electrotransformation protocol.
The data presented in this thesis represents the first report of the insertion of the streptococcal transposon Tn916 into the C. perfringens chromosome. Introduction of Tn916, harbored on the E. coli plasmid pAM120 delivery vehicle, was accomplished via electrotransformation. The transformation efficiency was 1.2 $\times$ 10$\sp2$ Tc$\sp{\rm r}$-C. perfringens transformants per $\mu$g of plasmid DNA. Tn916 has the potential for being exploited as a genetic tool for both insertional mutagenesis and cloning of specific clostridial gene sequences.
This study demonstrated that electroporation-induced transformation is an efficient, reproducible technique for the introduction of plasmid DNA into intact cells of different C. perfringens strains. The introduction of plasmid DNA molecules via this technique should allow for cloning and transposon mutagenesis studies of those C. perfringens genes involved in antibiotic resistance, and bacteriocin and toxin production.
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