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| − | Part Characterization
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| − | Materials and methods
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| − | E.coli TOP 10 strain transformation for part propagation
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| − | To transform BBa_K081005 4 µl of plasmid was incubated on ice for 30 minutes, heat-shocked for 2 minutes at 42°C in water bath. The cell wall was recomposed in test-tube in SOC medium 1ml for 3 hours.
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| − | Colonies were identified overnight after incubation at 37°C on solid LB medium with cloranfenicol as a screening method.
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| − | In order to transform BBa_K081005, 2 µl of plasmid was incubated on ice for 30 minutes, heat-shocked for 45 seconds at 42°C in a water bath, and later was put back on the ice again. The cell wall was recomposed on LB medium in eppendorf for 1 hour. Only a few colonies appeared after incubation for 36 hours at 37°C in solid LB medium with cloranfenicol as a screening method.
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| − | Miniprep
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| − | To extract both part's plasmidial DNA, a Cellco kit was used. Isolated TOP10 E.coli colonies were chosen from the previously obtained plates that were cultivated overnight in 5ml of LB medium with cloranfenicol. DNA was obtained from the selected colonies.
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| − | Digestion
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| − | BBa_K1499201 part was digested using 1.8 µl of DNA (554 ng/µl). Digestion was carried out with 1µl XbaI (NEB) and 1µl PstI (NEB), µl of NEBuffer 2.1, 1µl of BSA and 41. µl of MiliQ water. Incubation was carried out at 37°C.
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| − | BBa_K081005 was digested using 3µl of DNA(331 ng/µl), 1 µL of SpeI (NEB), 1µL of PstI (NEB), 5µl of NEBuffer 2.1, 1µL of BSA and 39 µl of MiliQ water. Incubation was carried out for 1h at 37°C.
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| − | After an electrophoresis, agarose gel extraction and purification was carried out with a PCR DNA extraction kit.
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| − | BioBricks ligation
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| − | The ligation step was carried out with 0.7 µL of digested BBa_K081005 as the backbone, 3.9 µL of digested BBa_K1499201 as insert, 2 µL buffer solution, 1 µL T4 Ligase (Thermo Fisher Scientific) and 12.4 µL MilliQ water. The reaction was incubated for 5 days at 18°C. The control consisted of a reaction without the insert and the same reagent volumes.
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| − | Transformation
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| − | Competent BL21 E.coli cells were transformed utilizing 20µL from the resulting solution from the ligation step. The transformation was incubated 30 minutes on ice, and the heat-shocked for 2 minutes at 42°C. Cell recomposition was carried out in eppendorf tube for 1 hour in 1ml SOC medium. Colonies were confirmed after overnight incubation in solid LB medium with cloranfenicol, at 37°C. Control reaction followed the same parameters, but without the plasmid.
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| − | Another transformation reaction was carried out, with the same parameters but with only BBa_K01005 as insert, this reaction resulted in E.coli that was posteriorly used as a control for the phenotypic test.
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| − | LB medium
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| − | The E. coli was cultivated in Luria-Bertani medium (LB), composed of tryptone1% (p/v), yeast extract 0,5% (p/v) and NaCl 0,5% (p/v). The pH was adjusted to 7,5. In order to make solid medium agar 2% was added.
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| − | Saline solution
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| − | For cell dilutions, 0.9% (w/v) NaCl solution was used.
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| − | Expression test
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| − | 2.5 mL of transformed bacteria were inoculated into 250 mL of LB medium and incubated at 37°C and 120 rpm. Bacteria without the plasmid were also inoculated and used as a control group. Then, 500 mL aliquots were taken from each of the strains (transformed and untransformed) at 1h, 2h, 3h, 3h30min, 4h, 4h30min, 5h, and 24h, counted from the incubation. To follow the bacterial growth rate, the optical density was also inferred at each period. Aliquots removed were centrifuged for 1 minute at 16000g. Then, the formed pellet was resuspended in freshly prepared running buffer (800 µl Loading Buffer 4x, 200 µl DTT). For the first point (1h period), 30 µl of buffer was used for resuspension. For the other points, 60 µl of buffer was used. Then the mixture was boiled for 5 minutes. Test analysis was performed using 15% SDS PAGE gel, applying equalized sample volumes.
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| − | Preparation of phenotype test plates
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| − | Transformed and control BL21 E. coli were inoculated into a test tube containing 2 mL of liquid LB medium. Incubation was carried out at 37°C, 150 rpm, for 24h. The extended incubation period was intended to guarantee that radiation tests would be performed with organisms in the stationary phase.
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| − | Plating
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| − | After irradiation of the samples, each irradiation point aliquot was inoculated in Petri dishes containing 20 mL of solid LB medium by a dripping technique. Plating was performed in triplicate and samples were diluted in 3 distinct concentrations. The plates were divided into 6 parts to hold the 3 distinct dilutions of each of the strains, as can be seen in figure 3.
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| − | Results
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| − | The expression test presented positive results for the expression of the desired protein. In figure 6, it is possible to visualize bands of expected size (27.7 kDa) in the transformed strain samples, while in the control group there is no band of the mentioned size. This demonstrates that D. radiodurans Uracil DNA glycosylase protein was successfully expressed by the strong constitutive promoter associated with a strong RBS. This provided support for phenotypic testing, with irradiation of samples at progressive doses of ultraviolet C radiation.
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