Production of bacteria colonies containing normal and mutated version of the same gene (encoding for a Bacillus thuringiensis’s toxin), in order to compare the activity of the corresponding proteins

Exposé Sciences & technologies Biologie

Date de publication :

05/12/2007

Langue :

French

Format :

.pdf

Nombre de pages :

13 pages

Niveau :

expert

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2 fois

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Sommaire :

Sommaire

Transfer of the gene of interest from pClone5 into a plasmid vector
1. Digestion of pClone5 and pEX
2. Electrophoresis
3. Excising the required bands from the gel
4. Purification of DNA from agarose slices
5. Ligation of purified DNA
Transformation of E.Coli with the ligated DNA
Confirming the presence of pEXClone5 in E.Coli cells
1. PCR
2. Rapid size screen
3. Electrophoresis
Site directed mutagenesis of pEXClone5
Analysis of mutant
1. Preparation of plasmid DNA
2. Digestion
3. Electrophoresis

Résumé :

The bacillus thuringiensis (Bt) is a bacterium which mostly has insects as hosts. When the growing conditions are bad (lack in nutrients, bad temperature etc.), Bt produces spores, sleeping forms of the bacterium, able to resist extreme conditions. What makes Bt different from other bacteria is its capacity to produce crystals during the sporulation. These crystals contain toxic proteins mostly directed against insects (used in agriculture). When the insect is dead, the spores use the nutrients thus released to grow again. Researchers have created many mutants to try to control the rate of toxins production within these bacteria. One mutant was found to produce more toxins, but it was growing a lot slower than the non-mutant bacteria. By creating plasmids from genomic DNA, one was found to complement the mutation: pClone5. The mutant that contained pClone5 was producing a normal amount of toxins and growing normally. The same gene was found in both mutant DNA and pClone5 plasmid, with a mutation on the 86th amino acid (a phenylalanine changed into a serine: in the codon sequence, a T had changed into a C). Using the bioinformatics tool we deduced that this gene encodes an adenylate kinase, enzyme catalysing the reaction 2ADPATP+AMP. Our project consists in preparing E.Coli colonies transformed with both non-mutated and mutated genes. This added to the deductions made on the protein structure (see appendix) should enable researchers to compare the activity of the mutated protein and the activity of the normal one. They will then be able to try to answer the following question: how does the mutation affect the activity of the protein, bearing in mind the peculiar phenotype conferred to Bt by the mutation. To do so, we will first transfer the gene from pClone5 into an expression vector pEX, resulting in the formation of the recombinant plasmid pEXClone5. Using a site-directed mutagenesis, we create the mutation on the recombinant plasmid. After having transformed E.Coli cells with pEXClone5 containing the mutation, we can see the activity of the mutated protein and compare it to the activity of E.Coli cells transformed previously with pEXClone5.