Sequencing verification of a domain exchange to identify the insect specificity region of the Bacillus thuringiensis israelensis toxin : an honors thesis (HONRS 499)

Abstract

Two of the Bacillus thuringiensis (B. t.) strains, israelensis and kurstaki, produce similar 130 kDa protoxins encoded by the crylVB and crylA(c) genes, respectively. The former encodes a toxin specific to dipteran larvae and the latter to lepidopteran insects. A putative insect specificity domain of cryl ti B was amplified by PCR and cloned into pUC 19 (pGI). This region was excised and exchanged with an analogous sequence in crylA(c) cloned in pKK2233 to form pOSM. The chimeric gene was stable in Escherichia coli but bioassays showed little toxicity to Aedes aegypti or Aedes triseriatus mosquito larvae. Sequencing of the inserts in both plasmids was performed to determine if there had been an alteration in the sequence resulting in loss of toxicity an/or to verify that we had amplified and exchanged the correct domain for toxicity. We verified that 33% of the insert contained no alterations. Toxicity may not have been seen due to the parental (pOSU4202 - B.t.k. cryIA(c) in pKK2233) losing toxicity.