Expression of the bacillus thuringiensis var. israelensis 130kDa delta-endotoxin and the firely luciferase reporter gene in escherichia coli

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dc.contributor.advisor Vann, Carolyn N. en_US Hicks, Teri A. en_US 2011-06-03T19:35:42Z 2011-06-03T19:35:42Z 1991 en_US 1991
dc.identifier LD2489.Z78 1991 .H53 en_US
dc.description.abstract The use of the larvacidal delta-endotoxin of the sporeforming bacterium Bacillus thuringiensis var. israelensis has been examined as a promising means to control insects that carry diseases such as malaria. An ultimate goal of this project was to genetically engineer both E. coli and the cyanobacterium Synechococcus PCC 7942 to express high levels of this delta-endotoxin and to construct the recombinant to carry a gene which would allow for monitoring of recombinants in the field. Previous research performed by a member of our laboratory involved cloning the gene fragment encoding the delta-endotoxin into a hybrid plasmid yielding recombinant E. coli clones which were toxic to mosquito larvae. Unfortunately, upon further examination of these recombinants using agarose gel electrophoresis and mosquitocidal assays, the clones were found to be unstable and lost their toxin encoding genes readily. Furthermore, cloning of the stabilizing parB locus into one of the recombinant plasmids did not enhance segregational stability as had been shown with some plasmids in E. coli. In another approach oligonucleotide primers were constructed which flanked the 130 kDa toxin gene but excluded a transposon-likesequence postulated to contribute to instability. These primers were used in the polymerase chain reaction in order to amplify this smaller DNA fragment for cloning experiments. Only a small quantity of primers were made and amplification of the DNA was not achieved prior to depletion of the primers. Future work will involve synthesizing new primers to be used for amplification and cloning of the B.t.i. toxin gene.In order to construct a traceable recombinant, the luciferase reporter gene (Luc) had been previously cloned into a hybrid plasmid that was capable of transforming both E. coli and the cyanobacterium Synechococcus PCC 7942. The new construction was then transformed into E. coli, to yield a pool of uncharacterized recombinants. In the present work, I determined that the luciferase enzyme was being expressed in the E. coli recombinants in the presence of the substrate luciferin. Initially, bioluminescence of these E. coli clones was detected by using OG-1 film which fogs in the presence of light. In order to quantify expression of the clones, lysates of the E. coli recombinants were also examined using a luminometer. Comparisons of bioluminescence were made between lysates with the parent E. coli plasmid harboring the luciferase gene and recombinants in which the Luc gene was placed downstream of the powerful rightward lambda promoter. Luminometer readings indicated that luciferase expression was enhanced six fold (from 2.0 X 10-6 to 3.0 X 10-5 by units/cell) in the recombinant plasmid. Plasmid DNA was isolated from the two luciferase expressing E. coli clones. Recombinants were obtained as determined by agarose gel electrophoresis examination of the plasmid DNA. This recombinant DNA was used to transform Synechococcus PCC 7942. However, because enzyme releasing methods were unsuccessful for the more rigid Synechococcus PCC 7942, the level of expression of the Luc gene could not be determined by either method mentioned above. Apparently, the methods used either failed to lyse the cells or they were too harsh and inactivated the enzyme. Future endeavors will involve the use of a French press to more gently lyse the cells so that the level of expression can be determined.
dc.description.sponsorship Department of Biology
dc.format.extent 62 leaves : ill. ; 28 cm. en_US
dc.source Virtual Press en_US
dc.subject.lcsh Insect pests -- Control. en_US
dc.subject.lcsh Escherichia coli. en_US
dc.subject.lcsh Bacillus thringiensis. en_US
dc.title Expression of the bacillus thuringiensis var. israelensis 130kDa delta-endotoxin and the firely luciferase reporter gene in escherichia coli en_US Thesis (M.S.)
dc.identifier.cardcat-url en_US

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  • Master's Theses [5256]
    Master's theses submitted to the Graduate School by Ball State University master's degree candidates in partial fulfillment of degree requirements.

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