TITLE: Preparation of a cell-free expression system from Escherichia coli
AUTHORS: Renuka Kudva

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the procedure make an S30 derived E.coli cell lysate that lacks membrane components. Modified from a protocol published in </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1091/mbc.e11-07-0590</span></a></div><div class = "text-block">The procedure takes three days in total, including preparation time. </div></div>

[STEPS]
?. Day 1: Make medium and autoclave 1. Wash all flasks with milliQ water.2. Prepare medium as described, autoclave. 4  erlenmeyer flasks, and one  flask with 50 ml medium for overnight culture. (This protocol uses S30 medium, see materials). 3. Autoclave milliQ water to make the buffers.4. Prepare stock solutions of all the buffers, filter sterilise. Prepare stocks of amino acids, PEP, Pyruvate Kinase, and ATP, and freeze at 5. Prepare S30 buffer ( and store at  ). 6. Set up pre-culture of E.coli cells (Top10 and MRE600 used in manuscript, protocol can be used for other strains too!). Inoculate either from a plate or from a glycerol stock of a clone which gave a functional cell extract. Grow pre-culture at  shaking at 200 rpm.
5 L
200 ml
4 L
?. Day 2: Ribosome isolation1. Set up main cultures () from the pre-cultures to an A600 of 0.1.2. Grow at   to an A600 of 1.0-1.2. (check growth every 20 min). 3. Cool down cells on ice for 4. Harvest cells by centrifugation at  for   in a JLA-8.1000 rotor (Beckman-Coulter).5. Resuspend cell pellet in  cold S30 buffer. Centrifuge again at . Repeat step 5 thrice. 6. Determine pellet mass. Resuspend in  S30 buffer/g cell mass. Plunge-freeze half of the suspension in liq Nitrogen and store at  .7. Supplement the other half of the cell suspension with 5 mM PMSF/1% Roche cOmplete protease inhibitor cocktail. 8. Lyse cells either through a french press or an emulsifex at 8000 psi. Three passes. 9. Clarify lysate by ultra-centrifugation. Twice for  in a Ti70 rotor (Beckman Coulter).10. Distribute clarified lysate into clean Ti70 polycarbonate centrifuge tubes to half its volume (around ), and       underlie with  sucrose cushion with a needle and syringe. (Use a 20G needle because the solution is viscous.)11. Centrifuge at  for   in an ultra-centrifuge (rotor Ti 70).
1 L
50 ml
1 ml
14 ml
14 ml
?. Day 3: Cell extract1. After 17 hours, decant the supernatant obtained after ultra-centrifugation and rinse the 70S pellet with  S30 buffer.  Store on ice. Shake occasionally by hand to resuspend gently. 2. Thaw the cells stored the previous day at  in an ice-water bath. 3. Lyse with a french press or emulsifex similarly to what was done for the ribosome cell pellet.4. Clarify lysate at  in TLA 100.3 tubes (Beckman Coulter) for . 5. Transfer the lysate  onto the ribosome pellet and homogenise with a dounce homogeniser (Wheaton, loose piston.)6. Transfer the cell extract to a  tube and treat the following way.      For each ml of lysate, add the following:   A160 ul Tris Acetate pH 7.520.6 ul 1 M DTT31.6 ul 1 M Magnesium acetate46 ul amino acid mix52 ul 0.25 M ATP (pH neutralised)627 ul 0.2 M phosphoenol pyruvate72.4 ul 2mg/ml pyruvate kinaseShake well (by hand) and incubate at 37°C for 1 hour. 7. Prepare a dialysis tube (pore diameter 14,000 to 16,000) by placing in beaker full of water and slow stirring for 30 min. 8. Transfer the cell extract into the dialysis tube and dialyse against 1 l S30 buffer thrice at 4°C. The third dialysis step can be carried out overnight. (This step can be adapted for use with PEG to concentrate the cell lysate).9. Aliquot the extract and flash freeze in liq Nitrogen. Store at -80°C long-term.
500 µl
15 ml
A160 ul Tris Acetate pH 7.520.6 ul 1 M DTT31.6 ul 1 M Magnesium acetate46 ul amino acid mix52 ul 0.25 M ATP (pH neutralised)627 ul 0.2 M phosphoenol pyruvate72.4 ul 2mg/ml pyruvate kinase