E.coli

E.Coli

E.Coli DH5α

It is a non-pathogenic strain of E.Coli that was developed for laboratory cloning use. The genomic structure of this strain is a singular circular chromosome consisting of 4,686,137 nucleotides, 4359 genes, and 4128 protein encoding genes. It has the ability to accept plasmid insertion exceptionally well and its several mutations enable high-efficiency transformations. [1] The different mutations dlacZ Delta M15 Delta(lacZYA-argF) U169 recA1 endA1 hsdR17(rK-mK+) supE44 thi-1 gyrA96 relA1 makes this strain an excellent strain for laboratory cloning procedures. The endA1 mutation allows for lower endonuclease degradation which ensures higher plasmid transfer rates and the ecA1 mutation reduces homologous recombination for a more stable insert. [2] Moreover, this bacterium reproduces by successive binary fission with a generation time of approximately 30 minutes with optimum growth occurring at 37 degrees centigrade. E. coli are facilitative aerobic bacteria and are capable of ATP synthesis via both aerobic respiration and, if oxygen is not present, fermentation. This particular strain can be identified and distinguished from other E. coli strains, by examining the genetic sequence of its 16s small ribosomal subunit, which has been fully sequenced. [1]

E.Coli BL21

It is a non-pathogenic strain of E.Coli that was developed especially for protein synthesis.

Choice of E.Coli DH5 α and E.Coli BL21

We chose to use Escherichia Coli strain DH5α as chassis for amplified our plasmid for its particular characteristics developed above. Indeed, this bacterium grows easily and has several mutations that make it an excellent choice for cloning procedures with a high efficiency transformation. All our parts are optimized for E. Coli. Moreover, E. Coli is the model organism, entirely sequenced.

Effect of COV and especially toluene on E.Coli

It is important to study the effect of pollutants on our chassis to better understand our results and predict the possible outcomes of those effects on the sensibility of our biosensor. It has to be known that toluene has been employed for many years by microbiologists to sterilize cultures and to maintain solutions in a sterile condition but also as an unmasking agent for several enzymes. Therefore, toluene has a deleterious effect on bacteria[3]. Toluene do not disrupt the cells nor denature its component. However, material lost from the cell treated with toluene was observed. Scientifics found that this loss of RNA (especially ribosomal RNA) and proteins was due to the complete and rapid destruction of the galactosidase permease as well as the disturbance of tryptophan permease activity. Others permeases activities seem also to be affected[3][4]. Moreover, toluene seems to elicit the enzymatic degradation of ribosomes followed by degradation of ribosomal RNA. Toluene treated cells also lose the ability to concentrate substrates and their macromolecule synthesis ability was affected. The toluene-treated cells were shown to exclusively synthesize membrane proteins[5]. As the respiratory chain of toluene treated cells seems to be intact, scientists establish that toluene do not trigger the total destruction of the membrane and that its lethal effects were mainly due to the loss of the ability to concentrate substrate, the loss of selective permeability and the loss of ribosomes. Toluene treated cells retains the machinery to provide itself with energy[3].

[1]https://microbewiki.kenyon.edu/index.php/DH5-Alpha_E.coli [2] Taylor, R. G., Walker, D. C. and McInnes, R. R. ( 1993). E. coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing. Nucleic Acids Res. 21, 1677 -1678. [3] Jackson, R.W. and Demoss, J.A. (1965). Effects of toluene on Escherichia coli. J. Bacteriol. 90, 1420–1425 [4] De Smet, M.J., Kingma, J., and Witholt, B. (1978). The effect of toluene on the structure and permeability of the outer and cytoplasmic membranes of escherichia coli. Biochimica et Biophysica Acta (BBA) - Biomembranes 506, 64–80. [5] Halegoua, S., Hirashima, A., Sekizawa, J., and Inouye, M. (1976). Protein Synthesis in Toluene-Treated Escherichia coli Exclusive Synthesis of Membrane Proteins. European Journal of Biochemistry 69, 163–167.