Transformed bacteria, which contain a plasmid with a target DNA, is placed into laboratory tube with 4 ml of liquid LB medium and specific antibiotic. Bacteria are then placed into the shaker during the night at 37ºC. For the plasmid DNA extraction we used GeneJet Plasmid Miniprep Kit, according to manufacturer's instructions.

The agarose gel is loaded into electrophoresis sytem, filled with the buffer. The DNA ladder and DNA samples are then loaded into the wells. Run the gel at 130V for 30-40 minutes.

Competent cells are centrifuged (2 min., 6000 rpm, 4 ºC). Supernatant is discarded, leaving about 100 µL of solution, in which cells are re-suspended. These cells are mixed with 10-20 ng of ligate or plasmid DNA. The mixture is incubated on ice for half an hour, then placed in a thermostat at 42 ºC for 1 minute. 1 mL of LB liquid medium is added to the mixture and cells are incubated in a shaking thermostat for 1 hour at 37 ºC. The tubes are pelleted by centrifuging; supernatant is discarded, leaving about 50 µL, in which cells are re-suspended. Transformants are placed onto a Petri dish with LB medium and strain-specific/vector-specific antibiotic. Plates are incubated in a thermostat at 37 ºC for 16 hours.

The cells are grown in 4 mL of LB medium for 2 hours in a shaking thermostat at 37 ºC. All the further steps are performed on ice. The cells are transferred into eppendorf tubes and pelleted by centrifuging for 2 minutes at 6000 rpm at 4 ºC. Supernatant is discarded and bacterial cells are re-suspended in 1 mL of NaCl solution. Tubes are centrifuged again for 2 minutes, 6000 rpm, 4 ºC. Supernatant is discarded and cells are re-suspended in 1 mL of CaCl2 solution. The tubes are left on ice for an hour or longer.

Transformed bacteria are grown overnight at 37 ºC in a 4 mL of LB medium and antibiotic. Next day, the overnight culture is transferred to the tubes with 20 mL LB medium at a dilution 1:20 and grown for 3 hours at 37 ºC. 0.2% arabinose is added to each tube. Bacteria are then grown at 37 ºC for 4-5 hours. Cells are pelleted and stored in -20 ºC or re-suspended in 1 mL Tris-NaCl buffer. 10 µL of PSMF is added to each tube. Cells are then sonicated and centrifuged for 20 minutes at 13200 rpm. Soluble fraction is transferred to the other eppendorf tube, while insoluble fraction is suspended in 1 mL of NaOH-SDS solution.

Two 12 % SDS protein electrophoresis gels are made - one for western bloting and one for SDS-PAGE. Each gel consists of separating (lower) and stacking (upper) gels. For the separating gel acrylamide solution, Tris-HCl (pH 8,8) buffer and milli-Q H2O is mixed with x μL APS and x μL TEMED solutions and poured into the gel preparation frame with the water on top of it. After that the stacking gel is made from acrylamide solution, Tris-HCl (pH 6,8) buffer, milli-Q H2O, x μL of APS and x μL of TEMED. Whole mixture is poured into the gel preparation frame (on top of the separating gel), comb inserted into the stacking gel and everything is left to gelate. Protein sample preparation: 45 μL of soluble and insoluble fraction samples after lysation are mixed together with 15 μL loading protein dye and denaturated for 10 minutes at 100°C. Prepared gel is put into gel loading machine, samples are loaded into the wells, as well as protein ladder. Reaction conditions are x V/cm voltage, gel is loaded for x minutes. After electrophoresis, one gel is placed in the transfer buffer, while the other is dyed with Page Blue dye.

Load SDS-PAGE and keep the gel in the transfer buffer after. The membraine and six pieces of Whatman paper are prepared: membraine is placed into methanol for a few seconds, then placed into transfer buffer; Whatman papers are also placed in transfer buffer for 10 minutes. Transfer from gel onto the membraine is performed in a western blot system for an hour, x mA. After transfer, membraine is blocked for half an hour in the blocking buffer. After blocking primary antibodies are placed in the buffer with the membraine and incubated for half an hour. After incubation, membraine is washed at least three times with wash buffer for 10 minutes. After washing, secondary antibodies are added to the buffer and membraine is incubated again for half an hour. After incubation, membraine wash is repeated as before. After washing membraine is visualized by adding it to substrate buffer together with 66 μL NBT and 33 μL BCIP and incubating for a few minutes.

Gel extraction was performed according to manufacturer's instructions.

PCR purification was performed according to manufacturer's instructions.

Turn on electroporator and optimize it for used bacteria strain. Place 1 mm electroporation cuvettes on ice. Add 1 µL of ligate/plasmid DNA solution to the cells in each of the microcentrifuge tubes. Transfer this mixture to the cold cuvette, tap on countertop two times, wipe water from exterior of cuvette and place in the electroporation module and press pulse. Add 1 mL of 37ºC LB medium, mix up by pipetting up and down and transfer to a 15 mL falcon tube. Shake in the incubator for one hour. Finally, place 10 µL of mixture on one LB-antibiotic plate, 100 µL on another and 200 µL on third and incubate them overnight at 37ºC.

All the reaction components are mixed on ice in specific proportions (adding polymerase last) and quickly transferred into preheated thermocycler.

All the reaction components, except the bacteria, are mixed on ice in specific proportions (adding polymerase last). To each PCR tube, containing the PCR reaction, the small amount of bacteria from colony is added. This is made with a wooden stick and each colony is marked on the plate, as well as transferred onto a new plate. The PCR tubes are quickly transferred into a preheated thermocycler.

All the reaction components are mixed on ice, adding the restriction enzymes last. Tubes are incubated at 37ºC for half an hour.

All the reaction components are mixed on ice, adding the ligase enzyme last. Tubes are incubated at room temperature for 10-15 minutes. Then ligase enzyme is inactivated by incubating tubes at 65ºC for 10 minutes. Afterwards, ligation reaction is chilled on ice and used in transformation.