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, which are able to resist even extreme conditions. What makes Bt different from other bacteria, is its capacity to produce crystals during sporulation. These crystals contain toxic proteins mostly directed against insects (used in agriculture). When the insect is dead, the spores use the nutrients released to grow again. Researchers have created many mutants to try to control the rate of toxin production within this bacteria.
[...] Moreover, we want to insert a restriction site of BamH1 and to do so, we will change the 84th amino acid changed into a T). Furthermore, we would like to trigger a mutation in the gene of resistance to ampicilline, to activate it. It allows us to check the site directed mutagenesis worked well because the L-Broth in which we put the cells contains ampicilline. Analysis of mutant Preparation of plasmid DNA Digestion Electrophoresis Results of the electrophoresis: This stage allows us to check that the mutagenesis worked well and thus that the cells of the culture have the mutation of interest. [...]
[...] We found out that grossly parts of the sequences are very similar between the same enzyme of different organisms. As we work on an enzyme, this specific sites well conserved might be catalytic or binding sites. The star on the alignment shown above indicates conserved sites: B.cereus Bt S.avermitilis M.musculus A.thaliana B.cereus Bt S.avermitilis M.musculus A.thaliana B.cereus Bt S.avermitilis M.musculus A.thaliana B.cereus Bt S.avermitilis M.musculus A.thaliana B.cereus Bt S.avermitilis M.musculus A.thaliana MAPNVLASE-----------PEIPK--------GIRAVLLGPPGAGKGTQAPKLAENFCV MATSSAASVDMEDIQTVDLMSELLRRMKCASKPDKRLVFIGPPGSGKGTQSPVIKDEFCL . : : : PHISTGDMFRAAMKAETEMGLQAKSFIDKGALVPDEVTIGIVRERLSQDDCVRGFLLDGF PHISTGDMIRAALKSGTEVGLQAKMFIDKGRLVPDELVIGIVRERISQDDCQRGFLLDGF PHISTGDLFRANISQQTELGKLAKSYMDEGNLVPDEVTIAMAKDRMEQPDAVNGFLLDGF CHLATGDMLRAMVASGSELGKKLKATMDAGKLVSDEMVVELIEKNLETPSCKNGFLLDGF CHLSTGDMLRAAVAAKTPLGVKAKEAMDKGELVSDDLVVGIMDEAMNRPKCQKGFILDGF : : * * : . [...]
[...] For the wells 3 and we can see two bands too, but the band for the plasmid migrated is less far because it is not digested. Thus, we can be sure that the cells digested contain a BamH1 restriction site and therefore that they contain the mutation of interest. Discussion In a nutshell, what we did is transforming E.Coli cells, some with gene encoding for the protein of interest and some others with the mutated gene. To do so, we first worked on pClone5, the plasmid which contains the gene of interest producing the normal protein. [...]
[...] In this way, we can select E.Coli cells which contain the desired mutation because they are also ampicilline resistant (see above procedure). The last step consisted of confirming the presence of the mutation in the cells. If the mutation has been successful, the plasmid should have two BamH1 restriction sites (as we created another one on the mutation). Therefore, the digestion by BamH1 followed by electrophoresis should present two bands. As we observed them, it worked. Now we have two types of cells, one able to synthesize the normal protein, and the other the mutated one. [...]
[...] This added to the deductions made on the protein structure (see appendix) enabling 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. [...]
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