May 13, 2020
Cell-free lysate (E. coli) preparation with sonication
- 1University of Edinburgh
Protocol Citation: Nadanai Laohakunakorn 2020. Cell-free lysate (E. coli) preparation with sonication. protocols.io https://dx.doi.org/10.17504/protocols.io.8nthven
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 23, 2019
Last Modified: May 13, 2020
Protocol Integer ID: 29107
Keywords: cell-free protein synthesis, synthetic biology, in vitro transcription translation
Abstract
Production of cell-free lysate from E. coli BL21 Star (DE3) with optional induction of T7 RNAP. Adapted from Kwon and Jewett 2015. Features:
- Variable starting culture sizes (10 mL - 1 L)
- Constant energy sonication
- S12 centrifugation
- Run-off and optional dialysis
CITATION
Protocol successfully used at the University of Edinburgh by Nadanai Laohakunakorn, and at EPFL by Barbora Lavickova and the 2017 iGEM team.
Materials
- Big centrifuge: Eppendorf 5810R with A-4-62 swing-bucket rotor (holds 50 mL tubes)
- Small centrifuge: Eppendorf 5424R with FA-45-24-11 rotor (holds 2 mL tubes)
- Vibra Cell 75186 sonicator / Qsonica Q125 + CL-18 probe
- spectrophotometer
- incubator with shaker
- autoclave
- magnetic stirrer
- LB/2xYTP/2xYTPG medium autoclaved
- E. coli strain of interest
- Tris base (Sigma T1503-100G)
- magnesium glutamate (L-glutamic acid hemimagnesium salt tetrahydrate) (Sigma T1503-100G)
- potassium glutamate (L-glutamic acid potassium salt monohydrate) (Sigma 49601-500G)
- DTT (1,4-dithiothreitol) (Sigma 10708984001)
- acetic acid
- deionized water
- autoclaved tips and petri dishes
- 500 mL Erlenmeyer flasks sterile
- culture tubes sterile
- 50 mL falcon tubes
- 2 mL eppendorf tubes
- 1 L beakers
- 10k MWCO dialysis cassettes (Slide-A-Lyzer, 3mL, Life Technologies)
- magnetic stir bar
- spectrophotometer cuvettes
- liquid nitrogen, dewar, -80 storage
Bacterial growth
Bacterial growth
Prepare all materials for bacterial culture
This protocol will make 200 mL of culture to yield around 1 mL of lysate with a protein concentration of around 40 - 80 mg/mL.
Reconstitute LB media from premix as per instructions on box.
Note
Protocol has also been successfully carried out with 2x YTP and 2x YTPG (34°C, original). Cells grow slower on YTP media (~5-6h to OD 1.5-1.8) compared to ~4h in LB.
Autoclave LB media for 00:20:00 at 121 °C along with tips and dishes
Grow overnight mini-culture
Add 5 mL of LB medium to a culture tube and inoculate with small amount of bacteria from glycerol stock.
Note
Strains used successfully with this protocol are
- BL21 (DE3) and BL21 star (DE3) (lacking RNaseE), T7 and E. coli RNAP
- Rosetta (DE3) for oscillators (contains rare tRNA encoding plasmid, good for eukaryotic protein expression)
- Top10, Top10-GamS (GamS inhibits RecBCD degradation of linear DNA), E. coli RNAP, alpha-complementation
- M15 (E. coli RNAP, alpha-complementation)
- DH5alpha (E. coli RNAP, alpha-complementation)
Grow overnight culture in an incubator at 37 °C and 200 RPM. Make sure cap is in loose position to allow for air exchange
Grow lysis culture (the next morning)
Measure and record OD600 of overnight culture at 10x dilution: pipette 900 µL LB and 100 µL overnight culture into a cuvette and take absorbance readings at 600 nm.
Expected result
OD600 should be around 4 (0.4 at 10x dilution)
Add 1 mL of overnight culture to 200 mL of LB medium in a 500 mL Erlenmeyer flask.
Incubate the culture for 04:00:00 , 37 °C , and 200 RPM.
Expected result
At the end of the step, the OD600 should be ~1.5-2. While Kwon and Jewett grow to OD600 = 3, we grow instead for a fixed time. They observe varying robustness of yield to final OD which is strain dependent. In the end we should probably grow to fixed OD.
Activity of cells harvested at different densities, grown in 1L 2xYTPG and sonicated at 1.5 mL/556 J. From Kwon and Jewett 2015, Figure 3, CC-BY license.
4h
(If required) induce after 2 hours with 0.4 mM IPTG (800 µL of 100 mM stock in 200 mL culture) to express T7 polymerase in the BL21 (DE3) strains.
A few minutes before incubation step finishes, prepare centrifuge and tubes
Cool down big centrifuge (Eppendorf 5810R) to 4 °C with fast temp mode; this takes around 10 mins.
Weigh one 50 mL Falcon tube along with its cap, and record. Clearly label the tube as well as its cap; this tube will be used to spin the final pellet.
As soon as the incubation finishes, put the Erlenmeyer flask On ice to arrest growth.
Centrifugation and cleaning
Centrifugation and cleaning
Put 50 mL of culture into each of four 50 mL Falcon tubes (one of which has been labelled), and spin at 4000 rpm , = 3220g 4 °C 00:20:00 . Tubes in swing-bucket rotor A-4-62.
20m
Put tubes back On ice ; keep bacterial pellet cold as much as possible.
Carefully discard supernatant using a pipette, then add 10 mL of Buffer A (no DTT) to each pellet. Resuspend by carefully pipetting up and down. Finally, transfer all four parts into the single labelled tube.
Balance centrifuge with a second tube containing 40 mL water. Then spin at 4000 rpm , = 3220g 4 °C 00:10:00 .
10m
Carefully discard supernatant using a pipette, then add 10 mL of Buffer A to the pellet. Then spin at 4000 rpm , = 3220 g 4 °C 00:10:00 .
10m
go to step #10 and repeat spin once more.
Finally, carefully discard as much supernatant as possible.
Weigh the tube containing the pellet (make sure cap is on), and record:
Final weight of tube + pellet
The wet mass of cells is the difference between this weight and the dry weight measured earlier. This is typically around ~1 g.
Flash freeze the cells using liquid nitrogen, respecting all safety procedures (wear protective glasses as there is danger of tube explosion etc.). Store the cells at -80 °C .
Note
Flash-freezing is optional (the protocol will work without) but it serves two purposes:
- Convenient pause point
- Frozen cells, once rethawed, lyse slightly more easily than fresh cells. So this step must be kept consistent to ensure reproducible protocol.
Sonication
Sonication
Keeping the pellet On ice , add Buffer A with DTT: 1 mL buffer per 1 g wet mass as determined earlier. This ratio is critical. The DTT must be prepared fresh as it will degrade otherwise.
Resuspend by vortexing, and put back On ice
Put 1 mL exactly of resuspended cells into a new 2 mL Eppendorf tube. This volume is critical.
Note
The larger the volume the more robust the lysis:
Yield as a function of volume and sonication energy. From Kwon and Jewett Figure 4, CC-BY license.
So if possible, scale up the production to achieve (ideally) at least 5 mL lysis volumes ( = 5 g wet mass).
Place the tube in an ice bath - ice mixed with water - and place the sonicator tip inside the tube so that it is immersed as much as possible but no part of the tip touches the tube surface. This is one of the most variable parts of the protocol. A suggested technique is to drop the tip to the bottom of the tube, and then raise it slightly. Holding the tube using a cool rack (e.g. Fisher 15592801) is recommended.
Sonicate until 400J has been achieved, using 50% amplitude and pulses of 10s : 10s (i.e. energy for 10s followed by pause for 10s). This typically takes around 00:01:24 but the time is variable. The total sonication energy is a critical parameter.
Separation and Clarification
Separation and Clarification
Cool down the small centrifuge (Eppendorf 5424R) to 4 °C using the fast temp mode: this takes around 20 minutes.
Balance and centrifuge the lysate at 12000 x g , = 11304 rpm 4 °C 00:10:00
10m
Transfer the supernatant to a new tube. To prevent any transfer of bacterial debris, it is critical that you do not take all the lysate.
Place the lysate in an incubator at 37 °C , 200 RPM for 01:30:00 , to degrade remaining DNA and RNA. This is called the run-off reaction.
Note
Even though Kwon and Jewett observe deterioriation in BL21 production yield with run-off, we do not see the same behaviour. If E. coli RNAP will be used, do dialysis as well as run-off.
1h 30m
Making sure the centrifuge is still cool, centrifuge the lysate at 12000 x g , = 11304 rpm 4 °C 00:10:00
10m
Transfer the supernatant to a new tube. Again, do not take all the lysate. Aliquot around 3 µL into separate tube for a Bradford assay.
(IF REQUIRED) carry out dialysis
Prepare Buffer A + DTT and add 900 mL to a 1 L beaker at 4 °C . Add a magnetic stir bar.
Rehydrate the required number of 10k MCWO cassettes in Buffer A+DTT in a beaker at 4 °C for 00:02:00
Load cassettes with 2.5 mL max (partial loading possible) of extract and dialyze for 03:00:00 f4 °C with stirring.
3h
Finally load extract into centrifuge and centrifuge the lysate at 12000 x g , = 11304 rpm 4 °C 00:10:00
Transfer the supernatant to a new tube. Again, do not take all the lysate. Proceed to step 27.
Keeping everything On ice , aliquot the lysate into small tubes as required (around 25 µL per tube is recommended).
Flash-freeze the remaining tubes in liquid nitrogen and store at -80 °C
To perform the Bradford assay, dilute 1 µL of the lysate in 99 µL of Buffer A. Mix 5 µL of this diluted lysate with 250 µL of Bradford reagent. Incubate for 00:05:00 before measuring the absorbance using a Nanodrop. The final protein concentration is typically 40 - 80 mg/mL.
Buffer A and B preparation
Buffer A and B preparation
Preparation of 1M stock solutions of tris acetate, magnesium glutamate, and potassium glutamate
Weigh 12.11 g of tris base (121.14 g/mol), add it to 100 mL of deionized water, and adjust to pH 8.2 with acetic acid, to make 1M tris acetate stock.
Weigh 38.86 g of magnesium glutamate (388.61 g/mol), and add it to 100 mL of deionized water, to make 1M magnesium glutamate stock.
Weigh 20.32 g of potassium glutamate (203.23 g/mol), and add it to 100 mL of deionized water, to make 1M potassium glutamate stock.
Preparation of 1L of buffer A
Add 10 mL of 1M tris acetate pH 8.2, 14 mL of 1M magnesium glutamate, and 60 mL of 1M potassium glutamate, and fill to 1 L with deionized water to make buffer A (10 mM tris acetate, 14 mM magnesium glutamate, 60 mM potassium glutamate).
Store at 4 °C
Add 2 µL of 1M DTT to 1000 µL Buffer A (final concentration 2 mM) for the lysis solution, or 1.8 mL of 1M DTT to 900 mL of Buffer A for the dialysis solution. This should be made fresh every time but if short-term storage is required, put at -20 °C
Citations
Kwon YC, Jewett MC. High-throughput preparation methods of crude extract for robust cell-free protein synthesis.
https://doi.org/10.1038/srep08663