Abstract:
The proposed study is concerned with the stabilization of expression of toxin genes in Escherichia coli and the cyanobacterium Synechococcus FCC 7442 which are specific for the larvae of Anopheles quadrimaculatus mosquitoes which may carry human malaria. The toxin genes are obtained from the soil bacterium Bacillus thringiensis subsp. israelensis (B.t.i.) which produces a crystalline endotoxin composed of insecticidal polypeptides. Clones have been constructed which contain part of the toxin gene in the hybrid vector pTNTV, which is able to transform both E. coli and cyanobacteria. Twelve of these clones produced significant toxicity to fourth instar larvae of a more susceptible mosquito species Aedis Aegypti. Ten of these twelve clones have recently been sty to have lost their B.t.i. insert due to segregational instability of the plasmid during replication. The plasmid pf(::Gi(:) Z contains a parB locus encoding a plasmid stabilization system which has been shown to stabilize E. coli and other gram negative bacteria. The primary goal of the proposed research is to insert this pqrB locus into the plasmid DNA of the vector pTNTV and the clones which still show toxicity, and to determine if this parB locus stabilizes expression of the toxin.
