Team:Vilnius-Lithuania/Methods

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Methods

Plasmid DNA extraction

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.

DNA electrophoresis

The agarose gel is loaded into electrophoresis system filled with the TAE buffer. The DNA ladder and DNA samples are then loaded into the wells. The gel is run at 120V for 30-40 minutes.

Bacterial transformation

Competent cells are centrifuged (2 min., 6000 rpm, 4ºC). 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 42ºC 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 37ºC. 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 37ºC for 16 hours.

Preparation of competent cells

The cells are grown in 4 mL of LB medium for 2 hours in a shaking thermostat at 37ºC. 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 4ºC. Supernatant is discarded and bacterial cells are re-suspended in 1 mL of NaCl solution. Tubes are centrifuged again for 2 minutes, 6000 rpm, 4ºC. 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.

Protein expression

Transformed bacteria are grown overnight at 37ºC 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 37ºC. 0.2% arabinose is added to each tube. Bacteria are then grown at 37ºC for 4-5 hours. Cells are pelleted and stored in -20ºC 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.

SDS-PAGE

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 17 µL APS and 10 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, 10 µL of APS and 10 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 100ºC. Prepared gel is put into gel loading machine, samples are loaded into the wells, as well as protein ladder. After electrophoresis, one gel is placed in the transfer buffer, while the other is dyed with Page Blue dye.

Western bloting

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

Polymerase chain reaction (PCR)

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

Colony PCR

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 transfered onto a new plate. The PCR tubes are quickly transfered into a preheated thermocycler.

Restriction

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

Ligation

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

PCR Purification

Gel extraction was performed according to manufacturer's instructions.

Gel extraction

PCR purification was performed according to manufacturer's instructions.

The evaluation of PAL activity

An overnight culture is inoculatated in 50 ml of LB medium (in a 500 ml Erlenmeyer flask) and grown at 30ºC and 200 rev min-1 for 12 h. After this, the grown culture is inoculated by 2% v/v in 50 ml LB medium in 500 ml Erlenmeyer flask and grown for 12 h at 30?C and 200 rev min-1. Later, 10 ml of culture is inoculated in 90 ml of fermentation medium in a 500 ml Erlenmeyer flask. Cells are incubated at 30ºC for 12 h (200 rev min-1) and grown for another 4 hours after induction in 42ºC.
Then, 10 ml of culture is centrifuged at 4000 x g for 6 min. Harvested cells are permeabilized by the treatment with Tween-20 or ethanol at the following concentrations: 0.15 g/l, 0.3 g/l, 0.45 g/l, 0.6 g/l for Tween-20 and 10%, 20%, 30%, 60%, 80% (v/v) for ethanol. Cells are resuspended in Tween-20 and ethanol solutions for 30 min in 30ºC. After this, cells are centrifuged and washed once with 25 mM Tris-HCl buffer (pH 8.8) (untreated culture, the cells are washed twice with 5 ml of 25 mM Tris-HCl (pH 8.8)
The reaction mixture of 5 ml containing 25 mM Tris-HCl (pH 8.8) and appropriate amount of L-Phe is incubated at 30ºC for 20 minutes. The whole reaction is arrested with 0.5 ml of 1 M HCl. After reaction cells are collected, centrifuged at 12,000 x g for 5 minutes and dried at 70-80ºC until dry cell mass is constant. After the deposition of cells, the reaction mixture is used to measure absorbance at 280 nm using. The concentration of trans-cinnamic acid in reaction mixture is then calculated by Beer-Lambert law.

Phenylalanine assay

Overnight culture is inoculated in 50 ml of LB medium in 500 ml Erlenmeyer flask and grown at 30ºC and 200 rev min -1 for 12 h. After this, the culture is inoculated by 2% v/v in 50 ml LB medium in 500 ml Erlenmeyer flask and left to grow for 12 h at at 30ºC and 200 rev min-1. Later, 10 ml of preculture is inoculated in 90 ml of fermentation medium in a 500 ml Erlenmeyer flask. Culture is incubated at 30ºC for 12 h (200 rev min-1), induced and supplied with an apprpriate amount of L-Phe and left to grow for 30 minutes at 37ºC. Phenylalanine concentrations from bacterial cultures were evaluated using Sigma-Aldrich Phenylalanine assay kit according to manufacturer's instructions.

Phenylalanine assay

1. The DNA is labeled with αP32-ddATP using TdT.

Incubate for 15 min at 37C, heat instantly for 5 min at 95C and put into ice after.

2. Prepare 15% denaturing acrylamide gel.

3. Mix the RNA samples with 2X RNA Loading Dye. Incubate for 5 min at 70C, instantly put into ice. 4. Electophoresis is ran at 1X MOPS buffer. First run the prephoresis for 20 min at 250V. Load the samples and run the electrophoresis at 150V for an hour. 5. RNA is transfered onto nylon membrane (7x9 cm) at 20V 250mA for 1h. 6. Prepare the hybridization buffer 7. Membrane is unpacked, placed in an empty clean bottle and washed 3 times with H2O. Then it is placed into the blotting chamber with the upper surface facing the glass. Incubate for 1h at 40C. 8. After one hour put 20 ul of the probe for hybridization at 40C for 12-18 hours. 9. Prepare wash buffer. 10. Membrane is washed three times with the wash buffer. 11. Membrane is put under a phospholuminescent screen to expose.

References

Influence of amino acids, organic solvents and surfactants for phenylalanine ammonia lyase activity in recombinant Escherichia coli, J.D. Cui, S.R. Jia, A.Y. Sun, Volume 46, Issue 6, June 2008, Pages 631–635
The "Ideal" Daily Intake of Threonine, Valine, Phenylalanine, Leucine, Isoleucine, and Methionine, Emanuel Cheraskin, M.D., D.M.D., W. Marshall Rmgsdorf, D.M.D., M.D., and Frances H. Medford, B.S., ORTHOMOLECULAR PSYCHIATRY, VOLUME 7, NUMBER 3, 1978, Pp. 150 -155

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