Expression and genetic manipulation of Bacillus thuringiensis toxins for improved toxicity and development of a protocol for in vivo selection of toxin variants with improved activity

George, Zenas Okon (2011) Expression and genetic manipulation of Bacillus thuringiensis toxins for improved toxicity and development of a protocol for in vivo selection of toxin variants with improved activity. Doctoral thesis (DPhil), University of Sussex.

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Abstract

Bacillus thuringiensis (Bt) and its insecticidal toxins have been used in pest
control for decades but there is a great concern about its future as a successful
pest control agent due to the development of insect resistance and the narrow
spectrum of activity of the toxins. To ensure the continuous relevance of Bt
toxins in pest control, projects aimed at isolating novel Bt strains expressing
toxins with improved activity are vigorously pursued and the genetic
manipulation of existing toxins to improve their activity and overcome resistance
is also undertaken. The aim of this project was to genetically manipulate the
genes encoding Cry1Ah and Cry1Ie for improved activity aimed at countering
resistance evolved by populations of Plutella xylostella. The project was also
aimed at expressing cry30Ea and cry40Da genes cloned from highly
mosquitocidal Bt strains S2160-1 and S2196 respectively and at developing a
protocol for the in vivo selection of toxin variants with improved activity. Cry1Ah
was successfully expressed in E. coli JM109 under the control of a cry1Ac
promoter and ribosome binding site and in Bt IPS/78/11 under the control of the
cyt1Aa promoter while Cry1Ie was also expressed in E. coli JM109. The
expressed Cry1Ah and Cry1Ie toxins were found to be toxic to both susceptible
(G88) and Cry1A resisitant (KARAK) populations of Plutella xylostella though
there was significant cross resistance to Cry1Ah in KARAK. A genetically
manipulated hybrid toxin CryAIA aimed at creating a novel toxin that captures
the relatively broad spectrum of Cry1Ah but overcoming KARAK resistance was
expressed but found to be non-toxic. Attempts to express cry30Ea and cry40Da
were also not successful despite utilising different hosts and expression vector
systems that have successfully been used in expressing other cry genes.
Meanwhile, the strategy designed to enrich for more toxic Bt strains in vivo in
from a mixed treatment in fact found that the non-toxic R128M strain dominated
the toxic 431 strain.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Depositing User: Library Cataloguing
Date Deposited: 27 Sep 2011 14:29
Last Modified: 17 Aug 2015 14:38
URI: http://sro.sussex.ac.uk/id/eprint/7392

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