Team:NFLS/Protocol
Transformation (heat shock)
1. Add 4 µl of the plasmid into BL21 cells that you have slowly defrosted on ice for 5 min and mix gently. Do not mix by pipetting up and down. Place SOC medium at room temperature.
2. Incubate on ice for 30 minutes.
3. Heat shock the cells for 30 seconds at 42°C without shaking.
4. Immediately transfer the tube to ice.
5. Add 250 µl of room temperature SOC medium.
6. Cap the tube tightly and shake the tube horizontally at 37°C for 30 minutes.
7. Spread 125 µl from the transformation on two selection plates by with a ‘spreader’.
8. Let the plates dry without the lid on.
9. Incubate the plates overnight at 37°C with agar upward and the lid downward.
Set up minipreps
1. Note the number of colonies on the plate.
2. Pick 1-8 colonies for analysis.
3. For each colony, inoculate in 3 ml LB containing 50 µg/ml antibiotics (kana) in a 11 ml tube using a 200 µl tip.Remember to assign a number to each tube/clone.
4. Grow overnight at 37°C with shaking (225-250 rpm).
Plasmid minipreparation
This protocol is for The GeneElute plasmid miniprep kit from Sigma Aldrich
For each miniprep culture:
Harvest & lyse bacteria.
1. Transfer 1.5 ml of the 3 ml overnight culture into a 1.5 ml eppendorf tube. Pellet cells from the eppendorf tube by 1 min centrifugation in the microcentrifuge (3000 x g). Discard supernatant.
2. Resuspend cells in 200 μl Resuspension Solution (cold). Pipet up and down.
3. Add 200 μl of Lysis Solution. Invert gently to mix. Do not vortex. Allow to clear for ≤ 5 min.
Prepare cleared lysate
4. Add 350 μl of Neutralization Solution (S3). Invert 4-6 times to mix.
5. Spin down the debris 10 min. at ≥12,000 x g.
Prepare binding column
6. Add 500 µl Column Preparation Solution to binding column in a collection tube.
7. Spin at ≥12,000 x g, 1 min. Discard flow-through.
Bind plasmid DNA to column
8. Transfer cleared lysate into binding column. (Important: be gentle at this step. Not to disturb the pellets that contain bacterial debris.)
9. Spin at ≥12,000 x g, 30 sec - 1 min. Discard flow-through.
Wash to remove contaminants
10. Add 750 μl Wash Solution to column. Spin at ≥12,000 x g, 30 sec - 1 min. Discard flow-through.
11. Spin 1 min. to dry column.
Elute purified plasmid DNA
12. Transfer column to a new collection tube.
13. Add 40 μl Elution Solution. Spin 1 min.
14. Check DNA concentration by choosing one smaple. Take 10 µl, add 90 µl of water and measure OD260. 1OD260= 50µg/ml DNA
15. Discard the column.
Store the DNA on ice until the next experiment (sequencing if needed).
Setting up overnight culture for protein expression
1. Transfer 1.5 ml of each culture to a new 11 ml tube and add 1.5 ml of LB medium containing 50 µg/ml antibiotics (kana).
2. Grow the culture at 37°C with vigorous shaking.
Recombinant protein expression
1. Transfer the clones to room temperature.
2. Add IPTG to a final concentration of 1 mM (3 µl of a 1 M stock per 3 ml) to both clones and grow the cultures at RT with shaking.
Protein purification
1. Transfer 1.5 ml of cells from each of the two selected cultures to a 1.5 ml tube, one for each culture. Spin the samples for 1 min at 3000 x g, and remove the supernatant. Repeat by transferring the remaining 1.5 ml of cells to the pellet. Spin the samples for 1 min, and remove the supernatant. These pellets are to be used to purify the recombinant protein.
2. Centrifuge the two tubes with Ni-NTA beads for 2 min at approximately 700 x g. Remove the ethanol buffer. Wash the Ni-NTA beads twice with 100 μl Buffer C in order to get rid of the ethanol by spinning 700 x g for 2 min each time.
3. Resuspend each pellet from step 1 in 600 μl Buffer B. Keep the samples on ice. Lyse cells by gently vortexing and treat the samples with ultrasound 4 times of 15 sec pulse with 10 sec break between each pulse. The tubes are kept on ice all the time during this procedure.
4. Centrifuge the lysates for 10 min at 15,000g to remove cellular debris, and transfer the supernatant to the tube containing the Ni-NTA beads.
5. The mixture of Ni-NTA beads and cell lysate is incubated for 30 min on ice. Vortex the tube every 5 min, or place the tube on a rotator.
6. Centrifuge the Ni-NTA beads/cell lysate mixture for 2 min at approx. 700g, and discard the supernatant.
7. Wash the Ni-NTA beads with 1000 μl Buffer C by inverting the tube several times (do not pipet) to resuspend and spin (2 min at approx. 700 x g). Remove the supernatant. This is done three times.
8. After the last wash remove the last buffer using a syringe and resuspend the beads in 25 μl Buffer E. Centrifuge for 2 min at approximately 700 g. Transfer the supernatant to a new 1.5 ml tube with a lid. Label it. Ad 25 μl Buffer S
Buffer B: 50 mM Tris pH 7.5, 0,5 M NaCl, 10 % glycerol, 0,1 % Igepal, 40 mM Imidazole, protease inhibitor tablet
Buffer C: 50 mM Tris pH 7.5, 0,5 M NaCl, 10 % glycerol, 0,1 % Igepal, 40 mM Imidazole
Buffer E: 50 mM Tris pH 7,5, 0,5 M NaCl, 10% glycerol, 0.1 % Igepal, 500 mM Imidazole
Buffer S: 50 mM Tris pH. 7.5, 200 mM NaCl, 20 % glycerol,
Buffer F; 50 % buffer E and 50 % buffer S
Screening Step Using the Congo Red Test
1. The cell strains were initially screened based on their ability to grow on a synthetic medium containing Avicel as the sole carbon source. The composition of the medium was as follows: NaNO3(3.0 g·L−1); K2HPO4 (1.0 g·L−1); MgSO4 (0.5 g·L−1); KCl(0.5 g·L−1); FeSO4·7H2O (0.01 mg·L−1); agar (20.0 g·L−1); Avicel (5.0 g·L−1). Only those strains that showed substantial growth in the initial screening with Avicel were selected for the Congo red test.
2. Inoculation was carried out by using a platinum needle to transfer the spores from the PDA plate to the center of the plates containing the CMC medium. The inoculated plates were incubated for 96 hat 30◦C and the growth of the microorganism was measured by the diameter of the colony.
3. A 10 mL aliquot of Congo red dye (2.5 g·L−1) was then added to each plate.
4. After 15 min, the solution was discarded and the cultures were washed with 10 mL of 1 mol·L−1 NaCl.
5. Cellulase production was indicated by the appearance of a pale halo with orange edges, indicative of areas of hydrolysis.
References
Correlation between Agar Plate Screening and Solid-State Fermentation for the Prediction of Cellulase Production by Trichoderma Strains. Enzyme Research Volume 2012.
