Induction of Protein Expression
Equipment & Consumables:
100 ml Sterile LB Liquid Media in baffled conical flask
Can scale this volume, ensure you scale the antibiotic and induction volumes linearly.
Aluminium Foil
5-15 ml Sterile LB Liquid Media in McCartney Bottles
5-15 µl Antibiotic Stock Solution
50 ml Falcon Tubes
OD600 Spectrophotometer
Cuvettes
Centrifuge (4000 rpm)
Induction Chemical at Desired Stock Concentration, e.g.
4 µl of a 100 mM stock of Cumate (Cumic Acid) (final concentration of 40 μM)
4-40 µl of a 100 mM stock of IPTG (final concentration of 40 or 400 µM) - varies depending on the protein being produced.
Plasmid map confirming antibiotic resistance and induction system
You probably should include controls in parallel with protein induction! This will double or triple your work, but will significantly improve troubleshooting;
Positive Control - Cells containing a well characterised plasmid with a similar induction system that can be used as a comparison.
Negative Control - Cells that contain the plasmid but are not provided the induction chemical. Alternatively you may use cells without the plasmid to obtain a baseline for the host.
Protocol:
Day 0: Fresh PLate of Desired Strain Containing Desired PLasmid
Ensure you have a relatively fresh plate of your desired expression strain of bacteria that contains your plasmid of choice.
NOTE: All pET15b plasmids will only work in BL21 E. Coli. Ensure your expression system is in the right strain if it is cell-specific.
If you only have plasmid material, do the heat shock protocol today and then inoculate from your heat shock plate tomorrow.
If you have plates that are older than 2 weeks, re-streak them onto a fresh plate and inoculate from this plate tomorrow.
Day 1: Seed Culture Inoculation
Label a sterile LB media in 15 ml McCartney Bottle with (plasmid-cell-media-antibiotic/name/date) e.g. CproX-DH5α-LB-Km/Alex/ 22/05
Using sterile technique, add 15 μl of antibiotic stock solution to your labelled 15 ml bottle.
Using sterile technique and an inoculation wand, pick up a well isolated colony from your fresh plate of bacteria and then gently stir the wand in the 15 mL McCartney bottle of LB media.
Feel free to flame the top for good luck.
Incubate overnight at 37°C (ideally with shaking)
Day 2: Culture inoculation and induction
Pre-heat the shaking incubator or water-bath to 37°C. It is good to pre-warm the Foil-capped baffled conical flask containing 100 ml of LB media as well, but not necessary.
Try to have an 18°C or RT shaker available as well, as you'll be swapping to a lower temperature after induction.
Label your Foil-capped baffled conical flask containing 100 ml of LB media with: (plasmid-cell-media-antibiotic/Inoculation Time: _______/Induction Time:_______ /name/date)
Using sterile technique, carefully lift the aluminium foil cap from the sterile conical flask containing LB media and extract and add 100 µl of correct antibiotic stock solution to your 100 ml of LB media. Replace the foil cap between steps.
Using the same careful sterile technique, extract 1 ml of sterile LB media with a pipette and transfer to a fresh Cuvette. Mark this cuvette "0" and use it to blank your spectrophotometer.
Using the same careful sterile technique, flame the top of the McCartney bottle containing the overnight seed culture and then pour the contents into the 100 ml baffled conical flask.
Ensure the aluminium foil cap is intact and not torn. If it is, try find something sterile to replace it or refold until the top is fully covered.
Move the conical flask to the 37°C shaking incubator (or water bath) and ensure it is secured fastly. Begin incubation with shaking at ~100 rpm (or as fast as you can go without breaking anything)
After 90 minutes, stop the shaker and remove the conical flask. Using sterile technique, carefully lift the aluminium foil cap from the sterile conical flask containing LB media and use a micropipette to extract 1 ml of inoculated media and add it to a fresh cuvette (labelled: 90). Replace the foil cap and return the conical flask to the shaking incubator.
Measure the OD600, likely it has barely changed. If it has changed noticeably, repeat the previous step after 30 minutes. Otherwise wait another hour to repeat OD600 measurement.
Repeat shaking incubation and measurement of OD600 at increasingly shorter intervals; keep in mind that once it reaches the exponential phase it can shoot past the desired 0.4-0.5 markvery quickly.
When the OD600 is between 0.4-0.5, it is time to induce the bacteria to start producing protein. Transfer the <100 ml culture in the baffled flask to an 18°C or RT shaker, secure it well and shake while it cools down for 10-15 minutes.
Optimal OD600 can vary between proteins, but start with this concentration and optimise later for your experiment.
Stop the shaker and remove the baffled conical flask. Using sterile technique, carefully remove the foil cap and add the required quantity of induction chemical.
4 µl of a 100 mM stock of Cumate (Cumic Acid) (final concentration of 40 μM)
4-40 µl of a 100 mM stock of IPTG (final concentration of 40 or 400 µM) - varies depending on protein, you can try optimise this step for your experiment.
Ensure the foil cap remains intact or make a fresh one.
Secure the culture back on the 18°C/RT shaker and shake for 6 hours or overnight.
Neither time is ideal, but at this point it's a really long day. Further investigation required to determine the exact point at which maximum yield is achieved. Good chance to go home and sleep while incubating.
Day 3 (Or 6+ hours later): Halt GRowth & Pellet
Label two 50 ml falcon tubes with (plasmid-cell-incubation time-incubation temp-name-date)
Record the total incubation time. Remove the foil cap from the flask and using a pipette-boi, distribute the completed batch of induced bacteria evenly between two 50ml falcon tubes.
Sterile technique is not strictly necessary at this stage but would be considered good practice.
Spin at 4000 rpm for 15 minutes in the large centrifuge. Pour off the supernatant into culture waste.
You can freeze the cell pellets for later, otherwise proceed to one of the cell lysis protocols.
Acknowledgements: