Creation of a stable and toxic Bacillus thuringiensis subsp. Israelensis recombinant plasmid vector within Escherichia coli and identification through mosquito larvae bioassay : an honors thesis [(HONRS 499)]

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McMillen, Patrick J.
Vann, Carolyn N.
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Thesis (B.?.)
Honors College
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Bacillus thuringiensis (B.t.), an extensively used microbial pesticide, produces protein endotoxins active against the larvae of many different orders of insects. The subspecies israelensis is specifically active against the larvae of certain mosquitos and blackflies which are known carriers of malaria. present use of the toxin involves the spraying of the crystal form of the toxin in the area of insect breeding, which is usually a large area of stagnant water. Frequent reapplications of the toxin are needed due to the toxin's sensitivity to ultraviolet light, and its tendency to sink and become unavailable for ingestion by the larvae (15).In the previous research done in the lab, the gene encoding the protein toxin from the BA A. bacterium was cloned into a plasmid capable of transforming bacteria, as well as cyanobacteria. The next step was to clone the plasmid into the cyanobacterium Svnechococcus POC 7942. This plan would alleviate some of the problems with spraying because the cyanobacterium would protect the endotoxin from ultraviolet breakdown and keep it in the feeding levels of the larvae. Esherirhia Eli was transformed and clones were identified. Bioassays were performed with the clones on the larvae of eedag aettvpti mosquitos and a few were shown to be toxic. Subsequent attempts to isolate the plasmid ttA from the clones, as well as subsequent attempts at bioassay showed that the Plasmid was unstable. Through studies done on the gene sequence, it was found that the gene may contain transposable sequences (17). A new approach was used to cut out the transposable sequences. Smaller, yet still active, gene =raO°eents were cloned into the target plasmid and E.coli clones have been identified. Bioassays have been done on a few of the clones and these show some t city to the Aed larvae. Two clones, #4 and #1, have shown significant ticity with the death of 3/5 of the exposed larvae. Further analysis is still being done to determine if there is stability.