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Cloning and expression in Escherichia coli of the insecticidal δ‐endotoxin gene of Bacillus thuringiensis var. israelensis
Author(s) -
Ward E.S.,
Ellar D.J.,
Todd J.A.
Publication year - 1984
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(84)80772-3
Subject(s) - bacillus thuringiensis , escherichia coli , cloning (programming) , gene , microbiology and biotechnology , biology , genetics , bacteria , computer science , programming language
Recombinant plasmids containing the mosquitocidal δ‐endotoxin gene were constructed by inserting Hin dIII fragments of the Bacillus thuringiensis var. israelensis 72–75 Md plasmid in to the Escherichia coli vector pUC12. Two recombinants producing the 26000 Da δ‐endotoxin (pIP173 and pIP174) were identified by screening clones in an E. coli in vitro transcription‐translation system. Both recombinants were 12.4 kb chimaeric plasmids comprising pUC12 and a common 9.7 kb Hin dIII fragment of the B . thuringiensis plasmid. The 26000 Da polypeptide synthesis in vivo from pIP174 transformed into E. coli JM101 was lethal to mosquito larvae and cytotoxic to mosquito cells in vitro. The biological authenticity of the cloned product was further confirmed by demonstrating that the cytotoxicity of the polypeptide was neutralised by antiserum to the authentic δ‐endotoxin or by preincubation with excess toxin receptor. Transcription of the recombinant δ‐endotoxin gene in E. coli appears to utilise a Bacillus promotor sequence(s) rather than the pUC12 β‐galactosidase promotor.