Bacillus thuringiensis subsp. israelensis (B.t.i.) produces a plasmid encoded parasporal crystalline protein which is larvacidal to mosquitoes carrying parasites for malaria and other infectious diseases. The purpose of this study was to construct a library of random fragments from the nine plasmids of wild type B.t.i. strain 402. The library was to be utilized in order to clone a 135kDa mosquitocidal polypeptide carried on a 108 kb B.t.i. plasmid.The library construction involved isolation of plasmid DNA by equilibrium density centrifugation, generation of random fragments of the nine plasmids by a partial Sau3A restriction digest, and ligation of these fragments into XbaI-BamHI restricted Lambda GEM-11 vector. Escherichia coli strain LE392 was infected by the packaged recombinant lambda and over 1000plaques were pooled to comprise the library. In order to verify construction of the library, both plaque screens of the library and Southern Analysis of restricted clones subjected to agarose gel electrophoresis were performed with labeled probes. The labeled probes were included: 1) radioactive end-labeled oligonucleotides constructed from published sequences of the B.t.i. 135 kDa toxic protein, 2) radioactive end-labeled random fragments from all nine plasmids of B.t.i., 3) radiolabeled entire plasmids of all nine plasmids of B.t.i., and 4) dioxigenin-labeled oligonucleotides. No homology between the lambda library digested DNA and the B.t.i. plasmid was observed. The results suggested that no lambda library of B.t.i. was constructed and, therefore, a lambda clone encoding the 135 kDa mosquitocidal polypeptide was not isolated. / Department of Biology
| Identifer | oai:union.ndltd.org:BSU/oai:cardinalscholar.bsu.edu:handle/183905 |
| Date | January 1990 |
| Creators | Litz, Sara Leandra |
| Contributors | Ball State University. Dept. of Biology., Vann, Carolyn N. |
| Source Sets | Ball State University |
| Detected Language | English |
| Format | iv, 54 leaves : ill. ; 28 cm. |
| Source | Virtual Press |
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