The Bioreactor suite houses bioreactors with culture volumes of 5L, 15L and 50L. You've probably worked with shaker flasks. As diving into the unknown can be rather intimidating, here's a quick summary of what to expect and do in the Bioreactor suite. The red items cover everything. Read the rest if you want to know more. The bottom line is:
Less work. More cells.
Advantages over shaker flasks
- Consistency – The entire volume can be exposed to the inducer at the desired OD, reducing the risk of variation between flasks.
- Temperature control – Water-jacketed bioreactors facilitate rapid cooling using chilled water to reduce and maintain a lower temperature during induction.
- High oxygen transfer rate – Air sparging combined with high agitation speeds increases the surface area of air in contact with the broth by creating many more smaller air bubbles. The resulting increased oxygen transfer rate from air to broth will support exponential growth to much higher biomass concentrations particularly if a richer medium is used (e.g. 2TY or Terrific Broth).
- Large biomass – Using richer media such as Terrific Broth or 2YT can support an OD of 20.0, reducing the volume of broth required to be processed. It might be possible to recover protein from a 5L bioreactor comparable to the amount yielded from 50L of LB. In the past, membrane protein yields from 5L TB were comparable to 15L fermentations of LB.
- Monitoring of parameters – Dissolved oxygen and pH are tracked over the course of the run. This not only indicating the progress of the fermentation but may also help optimize the process. Adjust blade speed based on that.
- Less work – You provide the inoculum and the chemicals. James will do the rest and return cell paste to you.
- Runs take between 14 and 16 hours. James will harvest the next morning after 10. Unless you express at 24°C or lower, this is not for you. However, the bioreactors can be programmed to cool to 10°C if cell growth needs to be slowed.
Your boss needs to include the Bioreactor suite in his/her risk assessment (Bio 1 form). For many groups, this is in place already. You'll need to supply James Mansfield with a grant code for charging. Note that your work has to meet containment level 1 requirements.
Doing a run
Being organized is critical. You'll need to email James to book a bioreactor for a week. Include the media recipe and the protocol for the fermentation in the email.
In the week of the run:
- On Monday, bring James ingredients to prepare the broth. Do your starter culture that same day.
- On Tuesday, provide inoculum (same proportion as used in shaker flasks), sterile-filtered antibiotics and inducer, and tubes to receive the cell paste.
- James will prepare broth, sterilize, inoculate, monitor OD, add inducer, harvest the cells, provide a run summary, and clean the bioreactor.
- On Wednesday, you pick up the cell paste.
As the bioreactor is yours for the week, there is some flexibility as to how the run is organized. Talk to James.
Rich media are recommended. If you're using LB, you're cheating yourself. TB is the simplest to prepare and good. Granules can be obtained from Sigma (T9179). Pouches to make 5L are cheap (£100 for two, Apr 2014) and most convenient. If you buy bulk, use 48 g per litre of broth. In both cases, supplement with 5 g/l glycerol.
An good alternative is 2YT where the presence of glucose suppresses leaky expression by T7. Some users swear by autoinduction media. Media is a parameter to optimize (see paper).
Here are two excellent recipes:
Supercharged Terrific broth
12 g/l tryptone
16 g/l tryptone
James will adjust pH to 7.4, autoclave and add 2 mM MgSO4.
James will autoclave.
M9 essential nutrients, vitamins, 10 µM FeSO4 (fresh) are optional.
Use kanamycin at 100 µg/l.
OD600 = 34 has been obtained.
The presence of glucose not only enriches the broth but also reduces leakage in T7 expression systems.
OD600 = 19 has been obtained.
Alternatively, you could use
- TSB – 17 g/l casein peptone, 3.0 g/l soya peptone, 5.0 g/l NaCl, 2.5 g/l HK2PO4, 2.5 g/l glucose (final pH 7.3 +/- 0.2 at 25°C)
- TB – 12 g bacto tryptone, 24 g bacto yeast extract, 5 g glycerol in 900 ml H2O, autoclave, add 100 ml filter-sterilized phosphate buffer (23.14 g/l KH2PO4, 125.41 g/l K2HPO4)
Optimization of yield
There are no rules for maximizing yield. Work out the best parameters before scaling up to bioreactors.
- Find the best broth, optimal induction temperature, cells, etc.
- Inducer concentration can have a big effect. Sometimes very low concentrations work much better.
- When expressing at low temperature, drop the temperature a doubling time before adding inducer.
For bioreactor cultures,
- avoid ampicillin. High cell densities mean lots of beta-lactamase in the broth. This can promote plasmid loss.
- keep pH between 6 and 8 for happy cells.
To avoid inclusion bodies, consider adding kanamycin to your broth one hour before harvesting (paper). This antibiotic targets the ribosome and shuts down translation, which gives the cellular folding machinery time to rescue aggregates from inclusion bodies. For this to work best, some protein should be in the soluble fraction in the expression test. With Rosetta cells (resistant to kanamycin), use another ribosome-targeting antibiotic like tetracyclin.
Optimization of inoculum
Ensure that your cells carry lots of plasmid. A quick way is to pick individual colonies and grow in separate 5 ml cultures. Use whichever grows fastest for a preculture overnight.
Inoculation and Growth
Inoculate a 0.5L preculture with one or two 5 ml cultures in the morning. Around lunchtime, hand preculture to James for inoculation. The OD of the preculture should be below 0.8 for happily growing cells.
- For induced growth, the culture temperature should be dropped prior to induction.
- For autoinduction, grow at 37°C from inoculation to late afternoon (~6h) and reduce temperature to 18-20°C and collect in the afternoon of the next day.