Development and application of a gene transfer system in Clostridium perfringens

Initially, electroporation-induced transformation of intact cells of C. perfringens 3624A-Rif$\sp{\rm r}$/Str$\sp{\rm r}$ with plasmids pAMβ1 and pHR106 resulted in transformation efficiencies of 1.4 $\times$ 10$\sp2$ and 1.2 $\times$ 10$\sp3$ transformants/μg 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 μg/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/μg DNA for plasmid plP401 to 9.2 $\times$ 10$\sp4$ transformants/μg DNA for plasmid pAK201. The greatest transformation efficiency obtained using pAK201 was 9.8 $\times$ 10$\sp6$ transformants/μg 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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Agriculture, Food Science and Technology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Allen, Steven Paul

Contributors dc:contributor

• Blaschek, Hans-Peter M.

• Agriculture, Food Science and Technology
• Biology, Microbiology

Statement dc:rights

• Copyright 1990 Allen, Steven Paul

Language dc:language

eng

Identifier

AAI9114159
(UMI)AAI9114159

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/23624