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Cloning of a Bacillus thuringiensis crystal protein gene

Olivia M. Nagy Arantes^I; Ghislaine Menou^II; Vincent Sanchis^II; João Lúcio de Azevedo^III; Didier Lereclus^II

^IDepartamento de Biologia Geral, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Campus Universitário, 86051 Londrina, PR, Brasil
IIUnité de Biochimie Microbienne, Institut Pasteur, 25 Rue du Dr. roux, 75724 Paris, Cedex, France
^IIIInstituto de Genética, Caixa Postal 83, Escola Superior de Agricultura "Luiz de Queiroz"/USP, 13400 Piracicaba, SP, Brasil. Send correspondence to J.L.A

ABSTRACT

The vector pUC18 and a shuttle vector containing the replication region of a resident plasmid of B. thuringiensis pHT3101, were used to clone a d-endotoxin gene of a newly isolated strain SPL407 (serotype H1). A 12 kb BamHI/Pstl fragment was isolated from total DNA cloned in E. coli using pUC18 as vector. A 12 kb Smal/PsI fragment was isolated from this construct and cloned into vector pHT3101 digested with SmaI/Pstl. A 7 kb Hpal/Smal fragment from this second construct was cloned into pHT3101 digested with Smal. The two latter plasmids were introduced into E. coli and B. thuringiensis. Analysis of plasmid DNA from the recombinant clones of B. thuringiensis indicated that it was not subject to molecular rearrangements and appeared structurally stable.

Keywords: Bacillus thuringiensis; Crystal protein.

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