1: Seed Culture, Inoculation, OD600 Monitoring, Induction & Growth
Introduction
Materials
- Micropipette and Sterile Tips
- Especially need recently sterilised 1mL Tips.
- Shaking Incubator or Shaking Water Bath
- Sterile LB Media (15mL) McCartney Bottles
- Foil-Capped Sterile LB Media (100mL) Baffled Conical Flask
- Aluminium Foil
- Kanamycin (50mg/mL)
- Ampicillin (100mg/mL)
- Cumate (Cumic Acid) (4-Isopropylbenzoic acid)
- Spectrophotometer
- Calibrate at start of day using: https://www.youtube.com/watch?v=20TpmeaTJho
- Cuvettes
- Tweezers to extract the cuvette from the spectro
- 50mL Falcon Tubes
- Centrifuge
Procedure
Day 1: Seed Culture
Day 0: 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. All pUS24X (aka. X) plasmids should work in a broad range of hosts.
- Label a sterile LB media in 15mL 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 15mL 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 15mL McCartney bottle of LB media.
Feel free to flame the top for good luck.
- Incubate overnight at 37°C (ideally with shaking)
Inoculation
Getting started early is recommended as the bacteria may be sluggish to grow at first, depending on how well the seed culture is going.
- Pre-heat the shaking incubator or water-bath to 37°C. It is good to pre-warm the Foil-capped baffled conical flask containing 100mL 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 100mL 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 100μl of antibiotic stock solution to your 100mL of LB media. Replace the foil cap between steps.
Kanamycin (50mg/mL) for Cumate Operon (X plasmids)
Ampicillin (100mg/mL) for T7 Operon (pET15b plasmids)
- Using the same careful sterile technique, extract 1mL of sterile LB media with a pipette and then move into a fresh Cuvette. Mark this cuvette "O" and use it to blank your spectrophotometer.
- Using the same careful sterile technique, flame the top of the McCartney bottle and then pour the contents into the 100mL 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
Ideal RPM? One that is fast but doesn't risk smashing the conical flask? More investigation required.
OD600 Monitoring
- After 1 hour, 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 1mL of inoculated media and add it to a fresh cuvette. Replace the foil cap and return the conical flask to the shaking incubator.
- Measure the OD600, likely at the 1 hour mark it has barely changed. If it has changed noticeably, repeat step 11 after 30 minutes. Otherwise wait another hour to repeat step 11.
- Repeat step 11 until the OD600 is above 0.3. At this point you should continue to monitor the OD600 at increasingly shorter intervals, it can shoot past the desired mark of 0.4-0.5 very easily.
- When the OD600 is between 0.4-0.5, it is time to induce the bacteria to start producing protein. If you are using a Cumate/pUS24X/X system, proceed to protocol (a). If you are using a pET15b-BL21/T7 expression system, proceed to protocol (b).
- Blank OD600 monitoring table:
A A | B B | C C | D D | |
1 1 | Time (mins) | OD600 | Plasmid-Cell: | |
2 2 | 0 | 0.00 | Inoculation Time: | |
3 3 | 60 | Induction Time: | ||
4 4 | 90 | Harvest Time: | ||
5 5 | a | |||
6 6 | b | |||
7 7 | c | |||
8 8 | d | |||
9 9 | e | |||
10 10 | ... |
a) Cumate Induction & Growth
For any cultures with a plasmid ending in X (e.g. CproX, PproX)
- Transfer the <100mL 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.
18°C is an impractical temperature to reach, but is considered 'ideal' for difficult to fold proteins. RT is fine and may even produce larger yields, but may result in more misfolding. Don't overthink it, just roll with RT for now.
- Stop the shaker and remove the baffled conical flask.
- Using sterile technique, carefully remove the foil cap and add Cumate (Cumic Acid) to a concentration of 40 μM. Ensure the foil cap remains intact or make a fresh one.
Do the calculation^
https://aem.asm.org/content/76/15/5058#T2 Increasing past 40 does not appear to help, but can go as high as 100μM
- 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. I recommend you go home and finish early tomorrow morning.
- (Either after 6-12 hours or early the next morning) Record the 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 cells for later, otherwise proceed to cell lysis protocol.
b) IPTG Induction & Growth
For any cultures with a pET15b plasmid (e.g. pET15b-Cpro) label. Occasionally these may be shortened to Cpro (without an X)
This expression system will only work in BL21 E. Coli. The plasmid must be grown in a different strain, purified and then heat shocked into BL21 within 2 weeks of the experiment.
- Transfer the <100mL 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.
18°C is an impractical temperature to reach, but is considered 'ideal' for difficult to fold proteins. RT is fine and may even produce larger yields, but may result in more misfolding. Don't overthink it, just roll with RT for now.
- Stop the shaker and remove the baffled conical flask.
- Using sterile technique, carefully remove the foil cap and add 4 or 40µL of a 100mM stock of IPTG (final concentration of 40 or 400µM). 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. I recommend you go home and finish early tomorrow morning.
- Label two 50mL falcon tubes with (plasmid-cell-incubation time-incubation temp-name-date)
- (Either after 6-12 hours or early the next morning) Record the 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 cells for later, otherwise proceed to one of the cell lysis protocols.
Set up a small seed culture to grow overnight. Come in early in the morning to Inoculate and then monitor the OD600. When the culture reaches the exponential phase of growth, induce it with either Cumate or IPTG.