WEEK 1

COMPETENT CELLS:

We prepared bacteria for the transformation of our plasmide of interest containing possibly the insert of interest. To be made, there is two methods: the chloride of calcium or by salts . We then transformed bacteria. The efficiency of the method of transformation can be deducted from the rate of transformation which can be calculated by making number of colonies by the 100 ng of plasmids added . It is recommended to use the same quantities of plasmids in every experiment of transformation to be able to compare the efficiency of the used method.

TRANSFORMATION:

We then spread the sample of bacteria transformed on petri dishes containing the antibiotic allowing the selection of bacteria having at least to integrate the plasmide. Colonies having grown the next day have all of the plasmide. A negative control is necessary for the comparison of the results, it is about a sample of bacteria having received no plasmide during the transformation.

EXPECTED RESULTS: bacteria not containing the plasmid, will not receive the gene of resistance in the conferred antibiotic. The not transformed bacteria are then going to die on the middle containing the kanamycin. OBTAINED RESULTS: Nevertheless, the next day we obtained some colonies on the cultures of the negative control. Sometimes, certain bacteria have naturally the resistance in the antibiotic of selection. This could indicate that it would be necessary to increase the doses of antibiotic in future experiments. We observed a higher rate of colonies having acquired the plasmide in the case of the method by salts rather than by chloride of calcium. What proves while the method by salts is more effective to return competent bacteria. In certain cultures few bacteria grew, it is due to the use of linear plasmids (digested) in the transformation. Bacteria contain only of circular DNA, the linear plasmid is then considered as foreign to the bacterium. By consequence, it is degraded thanks to the mechanisms of defense. However some colonies are obtained, this could be due to an imperfect plasmids digestion; in a spontaneous re-circularization of plasmids; either in re-circularization by the coverage of systems of repair of the DNA of the bacterium. COLONY PCR: A PCR colony is realized to test the presence of the insert in the plasmide acquired by the bacterium. We selected controls colonies, colonies transformed with the digested DNA and colonies transformed with the plasmidique not digested DNA (circular). Once realized, we realized a migration of the DNA on gel of electrophoresis, for the analysis of the product of PCR. EXPECTED RESULTS: size expected from the insert is 600 bases. In this case, we used the SacB system of identification of clones containing the insert in the plasmide: if SacB is amplified thanks to the primer 1 then the insert is not present in the plasmide. SacB is a very long DNA sequence, it will be then at the beginning of frost. If SacB is not then amplified,it is because there is an insert in the plasmide, and more particularly in the middle of the gene SacB. The insert will be then amplified by the primer 2. OBTAINED RESULTS: in every case, we obtained the plasmide and the primers. In the case of the controls, we were able to amplify SacB which is the highest band on the frost by its big size (see the well number 7 of Emeline). For other samples, we obtained a band in 600 bases which seems to correspond to our insert. In the case of certain samples, we sometimes obtain that the plasmide and the oligos what demonstrates the absence of insert in the plasmide of the clone. NOTICE: it would have been necessary to realize a negative control with a well of migration without matrix of DNA (without bacteria), it is the only condition of non-obtaining of bands. A positive control could also be in realized with a clone containing the cleansed plasmide.

MINIPREP:

in the following daytime, we realized Miniprep to extract the DNA plasmidique. After this stage of purification of the DNA, we measured the quantity of DNA thanks to the nanobot . Five positive clones for the presence of insert are used according to the results of the colony PCR, and a negative clone (without band at 600) is used as negative control. And then, we have to realize the third check, by digesting the DNA plasmidique cleansed. An analysis on frost of electrophoresis is made, to verify the presence of the insert. OBTAINED RESULTS: a single clone has proved to be good, by the obtaining of the band at 600 bases after digestion. Other clones then are to be eliminated. The positive clone could be used for the sequencing for finally that we are absolutely sure that the band at 600 bases is good the insert.

CHECK OF THE PRODUCTION OF A PROTEIN INDUCIBLE STEMMING FROM A PLASMIDE:

the last day, we made a check of overproduction of a protein of a system inductible, by the analysis on frost of agarose in 17 %, under the classic method of purification of proteins, before induction, after induction and with the method of the benzonase with or without induction. RESULTS OF THE WESTERN BLOT: on the Western Blot, we should obtain the band of the protein of interest only after induction, except us not induction of the production in case of the benzonase obtained the protein of interest with induction and, because there is always a flight of production of the protein even if she is not led by the molecular reactive of the promoter of the protein of interest. Furthermore, the use of the benzonase seems to increase the efficiency of migration in the frost. The benzonase degrades the DNA and seen that the protein of interest is a factor of transcription, her is bound to the DNA, and if the DNA is not degraded as in the case of the classic method, and good it prevents the good migration of the protein. Furthermore, the obtained bands are clearer, because the protein of interest better migrated. And it is also due to the stage of denaturation, the stage of TSTD in 95°C is more effective than 10 minutes in 37°C. BLUE: as regards the method of the Blue, we were able to observed the band of interest in the case only of the induction of the production of the protein. The benzonase seems increase the efficiency of the Blue to settle on protein for a better revelation of the studied protein of interest.