Preparation of E. coli
- Using a sterile plastic loop, pick 10-12 large (2-3 mm in diameter) colonies from the plate. Inoculate to 150 mL of SOB medium in a 1L flask, and grow overnight at 18-22°C, 200-250rpm. Alternatively, set up a starter culture (2 mL) overnight and inoculate the large scale in the morning. Grow at 37°C.
- OD600 should be 0.2-0.8 when harvested. Preferably, cells should be in mid log phase with OD600 ~0.5
- Remove the flask from the incubator and place on ice for 10 minutes. FROM THIS STEP, KEEP THE CELLS ON ICE AS MUCH AS POSSIBLE!
- Transfer the culture to a 15 mL centrifuge tube and spin at 2500 x g for 10 min at 4°C
- Pour off and discard the supernatant, and immediately place the tube on ice.
- Resuspend your cells in 1 mL of ice-cold TB buffer, make sure there are no clumps of cells left, but also treat your cells gently and keep them cold.
- Add ice-cold TB buffer to bring volume up to 1/5th of the original culture volume (~30 mL in this case). Mix the tube by gently inverting 3 times.
- Incubate the tube on ice for 10 minutes.
- Centrifuge at 2,500 x g for 7 minutes at 4°C, discard the supernatant.
- Gently resuspend the cells in ~1/20th of the original culture volume of ice-cold TB buffer. NOTE: 1/20th is based on an OD600 of 0.5, so adjust volume accordingly. E.g. if the culture OD600 was 0.1 then resuspend in 1/100th of original volume.
- Pre-chill 1.5 mL Eppendorf tubes on ice. Add 930 µL of your cell suspension, keeping the remainder on ice in the 15 mL tube.
- Add 70 µL of DMSO to the 930 µl of cell suspension. Mix gently by swirling, and place on ice.
- Aliquot 100 µL of the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the tubes with: Date – Strain. Snap freeze with liquid nitrogen or dry ice. Store cells at -80°C.
- Obtain competent cells from -80°C.
- Defrost, then return on ice immediately. Always keep cells on ice up till Step 4. Pipette 50 µL for each transformation. If you have 100 µL aliquot, split into 2 tubes.
- Add 2 µL of plasmid DNA/ ligation mix to each tube. Incubate on ice for 10-15 min.
- Heat shock in 42°C in block (or hot water in beaker with thermometer) for 45 seconds, then back on ice for 2 min.
- Recovery- Add 500 µL of SOC media to each tube, and incubate in the 37°C shaker for 90 min and shaking 500 rpm (recovery- 30min for plasmid or 90min for ligation mix).
- For each tube of cells, spread 20 µL onto one LB plate with appropriate antibiotic, and 200 µL onto a second plate, using aseptic technique. (Since colony counts have been so low we are currently only plating the 200 µL.) Remember to label your plate properly (Your name, sample name, cell line, antibiotic, date).
- Leave plates (with lid on) on bench or 37°C incubator to dry out before sealing with parafilm (or use cling wrap for multiple plates). Place your plate upside-down in the 37°C incubator.
- Harvest cells- Reserving 500 uL of culture for glycerol stock the remainder can be used for miniprep. Overnight culture - centrifuge 1 min to pellet cells. (Pour off supernatant and centrifuge more in the same tube. Keep quantities equal across your tubes to aid centrifuge balance. It is unnecessary to use all culture).
- Resuspend- 200 µL (this solution is refrigerated due to RNase).
- Lyse- 200 µL (gentle inversions < 5min) followed by precipitate/neutralize- 350 µL (gentle inversions).
- Centrifuge- 10 min (to pellet debris).
- Prepare columns- (cut lids off and label columns) 500 µL (column prep solution) - centrifuge 1 min.
- Collect DNA- pass lysate through column - centrifuge 1 min.
- Wash columns 750 µL - centrifuge 45 s (discard flow through) and centrifuge another 90 s.
- Elute DNA into labelled Eppendorf tubes. 30 µL - centrifuge 1 min. Retaining the elution and centrifuge through a 2nd time collects more DNA.
- Nanodrop to determine DNA concentration and label accordingly. (Use Miniprep elution solution as blank).
- Digest Upstream Part with EcoRI-HF and SpeI.
- Digest Downstream Part with XbaI and PstI.
- Digest the Destination Plasmid with EcoRI-HF and PstI: The Destination Plasmid should also have a different antibiotic resistance marker from both the plasmid containing the Upstream Part and the plasmid containing the Downstream Part to avoid the need to purify the Upstream and Downstream Parts.
- Incubate all 3 restriction digest reactions at 37°C for 60 min, then heat inactivate at 80°C for 20 minutes.
- Ligate the Upstream and Downstream Parts into the digested Destination Plasmid.
- Incubate the ligation mix at 16°C for 15 min, 30°C for 30 min, 37°C for 15 min, then heat inactivate at 80°C for 20 min.
- Digest the plasmid with EcoRI to create a linear plasmid and double digest with EcoRI and PstI to separate the gene and backbone (0.2 µL enzyme is sufficient for single or double digest no need to adjust).
- When making up a master mix consider making up a 2X concentration then the correct DNA volume to reach 100 ng with H2O added to balance.
- Incubate the two restriction digest reactions at 37°C for 30 minutes.
E. coli Hydrogen Production
|M9 Minimal salt (5X)||100 mL|
|RO water||400 mL|
Autoclave then add the following filter sterilised ingredients:
|Trace Elements||5 mL|
|1M MgSO4||0.5 mL|
|1M CaCl2||0.05 mL|
|1M MgSO4||0.5 mL|
|20% cas amino acid||2.5 mL|
|0.2 M Fe(NH4)SO4||125 µL|
|2% Thiamine||125 µL|
Top up to 500 mL with Sterile Milli-Q H2O.
- Add 2 mL of M9 media to a 15 mL tube with a colony of transformed E. coli for an overnight culture, 37°C and shaking.
- Measure the OD600 and dilute to get an OD600 of 0.1.
- Grow cells up to OD600 0.5.
- Induce with IPTG (1 mM) and add 20 mM glucose.
- Grow anaerobically for 24 hours, 37°C and shaking.
- Add induced culture at OD600 0.4 and dilute with M9 media supplemented with 20 mM glucose and 1 mM IPTG to give a final volume of 2 mL.
- Close tightly with plunger.
- Leave electrode to measure until the peak reaches its maximum and begins to drop, or until baseline is re-established.
- A large tub of water is filled. Three graduated measuring cylinders were then placed upturned with openings submerged into the tub of water. Flexible tubing was then attached to a bent glass tube fed into each cylinder.
- The other end of the tubing was attached to a Büchner flask with 80 mL mature culture in M9 media with an OD600 of 0.2, supplemented with 20 mM glucose and 1 mM IPTG.
- Flasks are stirred constantly and volume readings for each cylinder are taken at intermittent intervals.
- To test for the percent hydrogen gas in each headspace, a 20 mL gas sample was taken from each culture using a syringe and weighed.
- A sealed tube with 20 mL barium hydroxide was then put under negative pressure and the gas sample was injected and shaken for 10 minutes to remove CO2 in the form of barium carbonate, a stable precipitate.
- The remaining gas was extracted back into the same syringe and the volume recorded and again weighed (due to loss of volume and mass from CO2). The same volume of air was then weighed in the syringe, followed by the same volume of pure hydrogen gas.