subtilis challenged with low (LC) and high (HC) concentrations of total proteins of transformed E. Antimicrobial susceptibility testing results for apidaecin. With the lowest concentration of total proteins with apidaecin OD 600 decreases in 10.2% and with the highest one it decreases in 12.45%.įigure 6. At 21 h, both concentrations of apidaecin produced a decrease in OD 600 compared to the untransformed E. pyogenes (figure 2b) was treated with the same concentrations as B. However, both bacteria were inhibited in a certain percentage by this extract compared to the negative control of the total protein extract of the non-transformed bacteria. subtilis was found to be more susceptible to total protein extract with defensin 1 than S. However, the protein extracts of the bacteria transformed with the composite for the expression of defensin 1 showed greater inhibition, supporting the premise that the peptide is present and its activity is as expected. pyogenes, which allowed their inhibition. With the development of the antimicrobial susceptibility testing, it was observed that there was partial inhibition by both total protein extracts. coli BL21 (DE3) with defensin 1 and total proteins of untransformed E. pyogenes challenged with low (LC) and high (HC) concentrations of total proteins of transformed E. Antimicrobial susceptibility testing results for defensin 1. With the lowest concentration of total proteins with defensin 1 OD 600 decreases in 8.26% and with the highest one it decreases in 12.99%.įigure 5. At 21 h, both concentrations of defensin 1 produced a decrease in OD 600 compared to the untransformed E. pyogenes (figure 3b) was treated with the same concentrations as B.
#SNAPGENE SIMULATE AGAROSE GEL PLUS#
(On the right) Agarose gel electrophoresis of BBa_K2834006 compared with NEB Quick-Load® Purple 1Kb Plus DNA Ladder, where the highlighted band corresponds to approximately 2388 bp. (On the left) SnapGene® map of BBa_K2834006. (On the right) Agarose gel electrophoresis of BBa_K2834005 compared with NEB Quick-Load® Purple 1Kb Plus DNA Ladder, where the highlighted band corresponds to approximately 2442 bp.įigure 4. (On the left) SnapGene® map of BBa_K2834005.
(On the right) Agarose gel electrophoresis of BBa_K2834003 compared with NEB Quick-Load® Purple 1Kb Plus DNA Ladder, where the highlighted band corresponds to approximately 2336 bp.įigure 3. (On the left) SnapGene® map of BBa_K2834003.
Agarose gels allowed the confirmation of the correct plasmid constructions.įigure 2. Next step consisted of plasmid extraction and electrophoresis of the undigested plasmids, linearized plasmids with one enzyme, and double digested plasmids. Afterward, Escherichia coli BL21(DE3) cultures were transformed by heat shock for following antibiotic selection of clones. This resulted in complete expression plasmids of 2337 bp for apidaecin, 2442 bp for defensin 1, and 2388 bp for abaecin. Once the synthesis arrived, and the PCR was carried out, double digestion with EcoRI-HF and PstI restriction enzymes was made to each composite, and the chloramphenicol linearized plasmid backbone (pSB1C3) for following ligation of the backbone with each one of the fragments. The final parts resulted in a sequence of 310 bp for apidaecin, 415 bp for defensin 1, and 361 bp for abaecin. The three composites were synthesized by IDT® with the prefix and suffix flanking the region of interest.
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