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, the wells which contain the tube + and the tube are positive and negative controls and allow us to do a comparison: we expect to have in the wells 3 and 4 the same bands as the well but not the same as the well However, it doesn't work for the well because of a mistake of manipulation during the experiment (we think that we didn't take a colony with the toothpick). For the well we can in fact see two bands, the same as in well This colony thus contains pEXClone5 too. [...]
[...] We chose four organisms to work on: Bacillus cereus: 82% identities positives* Mus musculus: 44% identities positives Arabidopsis thaliana: 46% identities positives Streptomyces avermitilis: 52%identities positives *percentage of positives: the amino acids may not be the same but they have same properties Once we found these homologues, we worked on an alignment program (clustalW: www.ebi.ac.uk/clustalw/) to see whether they have similar sequences or not. We found out that grossly parts of the sequences are very similar between the same enzyme of different organisms. [...]
[...] ) KRLT cAMP and cGMP dependent protein kinase phosphorylation site STGD, SGTE, SQDD, SALE casein kinase II phosphorylation site (ser/thr kinase, independent for calcium) GLPGAG, GTEVGL N-myristoylation site (addition of myristate to N-term domain of eukaryotic proteins) TGR protein kinase C phosphorylation site (enhance the activity of PKC if phosphorylated) MNLV big-1 (bacterial Ig-like domain domain profile (allows mammalian host cells invasion by bacteria) FLLDGF/SPRTVAQ adenylate kinase signature (catalyses the reaction 2ADP ATP+AMP) (Mutation: phenylalanine changed for a serine) The motifs found are for some, impossible to find in a bacterium: our protein being extracted from bacteria, the protein is surely not able to do N-glycosylation, N-myristoylation, nor amidation. [...]
[...] To compare the activities of both mutated and non-mutated proteins, we want to create the mutation in some of these cells. To do so, we did a site directed mutagenesis of pEXClone5. Recall that pEXClone5 contains the mutated AmpR gene. We added to the template DNA two different primers: one triggers the wanted mutation on the gene of interest and the other complements the mutated sequence on the AmpR gene. By putting the single stranded template DNA and its primers into E.Coli cells, the DNA polymerase synthesizes the second strand and repairs the hybridizing errors caused by the primers that weren't totally complementary. [...]
[...] Another way of comparing the activities could be to compare the enzymatic activities of proteins, supplying the bacteria with the substrate ADP and recording the data. In this way, they could deduce how the mutation affects the enzyme, and particularly if it is the catalytic site which is affected or the binding site. We could also deduce whether the activity is enhanced or repressed or again cancelled or changed. Finally, we thought of another way of testing the activities: researchers could supply the mutants with many different substrates and compare the obtained phenotypes to the non-mutant one. [...]
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