TITLE: Cell-free lysate (E. coli) preparation with sonication
AUTHORS: Nadanai Laohakunakorn

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Production of cell-free lysate from E. coli BL21 Star (DE3) with optional induction of T7 RNAP. Adapted from Kwon and Jewett 2015. Features:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Variable starting culture sizes (10 mL - 1 L)</li><li style = "counter-reset:ol0;list-style-type:disc;">Constant energy sonication</li><li style = "counter-reset:ol0;list-style-type:disc;">S12 centrifugation</li><li style = "counter-reset:ol0;list-style-type:disc;">Run-off and optional dialysis</li></ul></div><div class = "text-block">Protocol successfully used at the University of Edinburgh by Nadanai Laohakunakorn, and at EPFL by Barbora Lavickova and the 2017 iGEM team.</div></div>

[STEPS]
?. [Bacterial growth]
Prepare all materials for bacterial cultureThis protocol will make  of culture to yield around  of lysate with a protein concentration of around 40 - 80 mg/mL.
200 ml
1 ml
?. [Bacterial growth]
Grow overnight mini-culture
?. [Bacterial growth]
Reconstitute LB media from premix as per instructions on box.
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.
?. [Bacterial growth]
Add  of LB medium to a culture tube and inoculate with small amount of bacteria from glycerol stock.
5 ml
Strains used successfully with this protocol areBL21 (DE3) and BL21 star (DE3) (lacking RNaseE), T7 and E. coli RNAPRosetta (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-complementationM15 (E. coli RNAP, alpha-complementation)DH5alpha (E. coli RNAP, alpha-complementation)
?. [Bacterial growth]
Grow overnight culture in an incubator at  and 200 RPM. Make sure cap is in loose position to allow for air exchange
37 °C
?. [Bacterial growth]
Autoclave LB media for  at   along with tips and dishes
121 °C
?. [Bacterial growth]
Grow lysis culture (the next morning)
?. [Bacterial growth]
Add  of overnight culture to  of LB medium in a 500 mL Erlenmeyer flask.
1 ml
200 ml
?. [Bacterial growth]
Measure and record OD600 of overnight culture at 10x dilution: pipette  LB and  overnight culture into a cuvette and take absorbance readings at 600 nm.
900 µl
100 µl
OD600 should be around 4 (0.4 at 10x dilution)
?. [Bacterial growth]
Incubate the culture for  ,  , and 200 RPM.
37 °C
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.
?. [Centrifugation and cleaning]
Put  of culture into each of four 50 mL Falcon tubes (one of which has been labelled), and spin at  . Tubes in swing-bucket rotor A-4-62.
50 ml
Centrifuge: 4000 33
4 °C
?. [Centrifugation and cleaning]
Put tubes back  ; keep bacterial pellet cold as much as possible.
on ice
?. [Bacterial growth]
A few minutes before incubation step finishes, prepare centrifuge and tubes
?. [Bacterial growth]
Cool down big centrifuge (Eppendorf 5810R) to  with fast temp mode; this takes around 10 mins.
4 °C
?. [Bacterial growth]
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.
?. [Bacterial growth]
As soon as the incubation finishes, put the Erlenmeyer flask  to arrest growth.
on ice
?. [Centrifugation and cleaning]
Carefully discard supernatant using a pipette, then add  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.
10 ml
?. [Centrifugation and cleaning]
Balance centrifuge with a second tube containing  water. Then spin at  .
40 ml
Centrifuge: 4000 33
4 °C
?. [Centrifugation and cleaning]
Carefully discard supernatant using a pipette, then add  of Buffer A to the pellet. Then spin at  .
10 ml
Centrifuge: 4000 33
4 °C
?. [Centrifugation and cleaning]
and repeat spin once more.
?. [Centrifugation and cleaning]
Finally, carefully discard as much supernatant as possible.
?. [Centrifugation and cleaning]
Weigh the tube containing the pellet (make sure cap is on), and record:   A1The wet mass of cells is the difference between this weight and the dry weight measured earlier. This is typically around ~1 g.
A1
?. [Centrifugation and cleaning]
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
Flash-freezing is optional (the protocol will work without) but it serves two purposes:Convenient pause pointFrozen cells, once rethawed, lyse slightly more easily than fresh cells. So this step must be kept consistent to ensure reproducible protocol.
?. [Sonication]
Keeping the pellet  , add Buffer A with DTT:  buffer per  wet mass as determined earlier. This ratio is critical. The DTT must be prepared fresh as it will degrade otherwise.
on ice
1 ml
1 g
?. [Sonication]
Resuspend by vortexing, and put back
on ice
?. [Sonication]
Put  exactly of resuspended cells into a new 2 mL Eppendorf tube. This volume is critical.
1 ml
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).
?. [Sonication]
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.
?. [Sonication]
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  but the time is variable. The total sonication energy is a critical parameter.
?. [Separation and Clarification]
Cool down the small centrifuge (Eppendorf 5424R) to  using the fast temp mode: this takes around 20 minutes.
4 °C
?. [Separation and Clarification]
Balance and centrifuge the lysate at
Centrifuge: 12000 34
4 °C
?. [Separation and Clarification]
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.
?. [Separation and Clarification]
Place the lysate in an incubator at  , 200 RPM for  , to degrade remaining DNA and RNA. This is called the run-off reaction.
37 °C
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.
?. [Separation and Clarification]
Making sure the centrifuge is still cool, centrifuge the lysate at
Centrifuge: 12000 34
4 °C
?. [Separation and Clarification]
Transfer the supernatant to a new tube. Again, do not take all the lysate. Aliquot around  into separate tube for a Bradford assay.
3 µl
?. [Separation and Clarification]
Keeping everything , aliquot the lysate into small tubes as required (around  per tube is recommended).
on ice
25 µl
?. [Separation and Clarification]
Flash-freeze the remaining tubes in liquid nitrogen and store at
-80 °C
?. [Separation and Clarification]
To perform the Bradford assay, dilute  of the lysate in  of Buffer A. Mix  of this diluted lysate with  of Bradford reagent. Incubate for  before measuring the absorbance using a Nanodrop. The final protein concentration is typically 40 - 80 mg/mL.
1 µl
99 µl
5 µl
250 µl
?. [Buffer A and B preparation]
Preparation of 1M stock solutions of tris acetate, magnesium glutamate, and potassium glutamate
?. [Buffer A and B preparation]
Weigh  of tris base (121.14 g/mol), add it to  of deionized water, and adjust to pH 8.2 with acetic acid, to make 1M tris acetate stock.
12.11 g
100 ml
?. [Buffer A and B preparation]
Weigh  of magnesium glutamate (388.61 g/mol), and add it to  of deionized water, to make 1M magnesium glutamate stock.
38.86 g
100 ml
?. [Buffer A and B preparation]
Weigh  of potassium glutamate (203.23 g/mol), and add it to  of deionized water, to make 1M potassium glutamate stock.
20.32 g
100 ml
?. [Buffer A and B preparation]
Preparation of 1L of buffer A
?. [Buffer A and B preparation]
Add  of 1M tris acetate pH 8.2,  of 1M magnesium glutamate, and  of 1M potassium glutamate, and fill to   with deionized water to make buffer A (10 mM tris acetate, 14 mM magnesium glutamate, 60 mM potassium glutamate).
10 ml
14 ml
60 ml
1 L
?. [Buffer A and B preparation]
Store at
4 °C
?. [Buffer A and B preparation]
Add  of 1M DTT to  Buffer A (final concentration 2 mM) for the lysis solution, or   of 1M DTT to  of Buffer A for the dialysis solution. This should be made fresh every time but if short-term storage is required, put at
2 µl
1000 µl
1.8 ml
900 ml
-20 °C
?. [Bacterial growth]
(If required) induce after 2 hours with 0.4 mM IPTG (  in  culture) to express T7 polymerase in the BL21 (DE3) strains.
[of 100 mM stock]
200 ml
?. [Separation and Clarification]
(IF REQUIRED) carry out dialysis
?. [Separation and Clarification]
Rehydrate the required number of 10k MCWO cassettes in Buffer A+DTT in a beaker at  for
4 °C
?. [Separation and Clarification]
Load cassettes with  of extract and dialyze for  with stirring.
[max (partial loading possible)]
4 °C
?. [Separation and Clarification]
Finally load extract into centrifuge and centrifuge the lysate at
Centrifuge: 12000 34
4 °C
?. [Separation and Clarification]
Transfer the supernatant to a new tube. Again, do not take all the lysate. Proceed to step 27.
?. [Separation and Clarification]
Prepare Buffer A + DTT and add  to a 1 L beaker at  . Add a magnetic stir bar.
900 ml
4 °C