African Journal of Biotechnology Vol. 4 (7), pp. 580-585, July 2005          
ISSN 1684–5315 © 2005 Academic Journals

Full Length Research Paper

Determination of the plasmid size and location of d-endotoxin genes of Bacillus thuringiensis by pulse field gel electrophoresis

Roderick L. Rolle*, Anthony O. Ejiofor, Terrance L. Johnson

Biological Science Department, Tennessee State University 3500 John A. Merritt Blvd. Nashville, TN. 37209-1561^a         

*Corresponding author. E-Mail: rrolle@mytsu.tnstate.edu.

Accepted 16 May, 2005

The genes encoding the d-endotoxins of Bacillus thuringiensis are located on plasmids ranging in size from 45 to 1000 kb.  Plasmid size and variety are diagnostic features for characterizing subspecies of this aerobic spore-forming crystalliferous entomopathogen. Two of 25 B. thuringiensis isolates obtained from Middle Tennessee were characterized into subspecies on the basis of size, number, and varieties of plasmids they host using pulse field gel electrophoresis (PFGE).  By using specific pulse angle (120°), field strength (5 V/cm), pulse time (26 h), and agarose concentration (1.2%) these DNA molecules were separated from other genomic DNA. The purified DNA product the agarose gel was tested for homology to 49 d-endotoxin gene using PCR.. The PFGE data for 5 of the 25 isolates collected in Tennessee showed distinct banding patterns. Two of the isolates had only 1 band whereas the others had more than 4 ranging from 45 to 1000 kb in size. Isolate 6 and 10 both yielded one 49 kb fragment that contained the cry1A gene.

Key words: Bacillus thuringiensis, d-endotoxin, pulsed-field gel electrophoresis, plasmid.