Editing S30 E. coli extract (section)

== Protocol 1, S30 E. coli extract ==

The amounts here are based on 1 L of culture which can be grown<br/>in a single Tunair 2.5-L shake fl ask (IBI Scientifi c). Using 2YTPG<br/>medium and the A19 strain, each liter of culture should produce<br/>about 8 g wet cell weight (harvested at 3 OD) which will yield<br/>about 10 mL of extract. The amounts can be scaled up if more<br/>extract is needed. In that case, it may be more convenient to grow<br/>the culture in a fermenter or bioreactor.

2.1. Cell Growth and Harvest<br/>1. E. coli strain A19 ( see Note 1 ).<br/>2. 2.5-L Tunair FUL-BAF fl ask with a Dri-Gauze fi lter lining in the cap ( see Note 2 ).<br/>3. 2YT medium for inoculum, 50 mL: 16 g/L tryptone, 10 g/L yeast extract, 5 g/L sodium chloride ( see Note 3 ).<br/>4. 2YTPG medium ( 2 ) , 1 L: 16 g/L tryptone, 10 g/L yeast extract, 5 g/L sodium chloride, 22 mM sodium phosphate<br/>monobasic, 40 mM sodium phosphate dibasic, 100 mM glucose, 100 m L antifoam 204 (Sigma-Aldrich), optional ( see<br/>Note 4 ).<br/>5. 1 M potassium, sodium, or ammonium hydroxide for pH control, optional ( see Note 5 ).<br/>6. S30 buffer, approximately 50 mL (about 150 mL total needed for the complete process): 10 mM Tris acetate, 14 mM<br/>magnesium acetate, 60 mM potassium acetate (see Note 6).

<br/>2.2. Biomass Processing<br/>1. S30 buffer, approximately 100 mL: 10 mM Tris acetate, 14 mM magnesium acetate, 60 mM potassium acetate ( see Note 6 ).<br/>2. Avestin Emulsifl ex homogenizer.<br/>3. Pre-incubation mix, about 3 mL: 370 mM Tris acetate pH 8.2, 11.1 mM magnesium acetate, 16.5 mM ATP, 50 m M each of<br/>the 20 amino acids, 105 mM phosphoenol pyruvate (PEP), 8.4 U/mL pyruvate kinase (Sigma-Aldrich #P7768).

2.3. Extract Testing<br/>1. 10× salt solution: 1.3 M potassium glutamate, 100 mM ammonium glutamate, 80 mM magnesium glutamate ( see Note 7 ).<br/>2. 10× master mix: 12 mM ATP, 8.5 mM GTP, 8.5 mM CTP, 8.5 mM UTP, 340 m g/mL folinic acid, 1.7 mg/mL E. coli total tRNA (Roche).<br/>3. AA mix: 50 mM each of the 20 amino acids ( see Note 8 ).<br/>4. 1 M sodium pyruvate.<br/>5. 1 M sodium oxalate.<br/>6. 200 mM putrescine.<br/>7. 200 mM spermidine.<br/>8. 20 mM coenzyme A (CoA).<br/>9. 100 mM nicotinamide adenine dinucleotide (NAD).<br/>10. L -[ 14 C(U)]-leucine (PerkinElmer).<br/>11. T7 RNA polymerase ( see Note 9 ).<br/>12. Plasmid DNA ( see Note 10 ).<br/>13. Whatman 3MM chromatography paper.<br/>14. Straight pins.<br/>15. 5% (w/v) trichloroacetic acid (TCA).<br/>16. Ethanol (95–100%).<br/>17. Scintillation cocktail.<br/>18. Liquid scintillation counter.

<br/>1. Inoculate 50 mL of 2YT medium in a 250-mL baffl ed fl ask with at least 10 m L from a thawed glycerol stock of E. coli A19.<br/>Alternatively, inoculate from a colony on an agar plate or a tube culture. Incubate the culture overnight at 37°C with<br/>vigorous shaking (~250–280 rpm).<br/>2. In the morning, transfer the entire 50 mL culture into 1 L of 2YTPG medium in a 2.5-L Tunair FUL-BAF fl ask with a Dri-<br/>Gauze fi lter lining in the cap ( see Note 2 ). Incubate at 37°C vigorous shaking (~250–280 rpm) and monitor growth by<br/>OD at 600 nm.<br/>3. Harvest the culture during exponential phase before the growth rate drops during the transition to stationary phase<br/>( see Note 11 ). The culture should be chilled as quickly as possible either by adding ice directly to the culture or by passing it<br/>through a heat exchanger or cooling coil in ice water.<br/>4. Once the culture is chilled, collect the cells by centrifugation at 8,000 × g for 20–30 min. If the entire culture volume cannot<br/>be centrifuged at once, pour the supernatant off the cell pellets and add more culture on top the pellet for a second centrifugation.<br/>Approximately 2.5 g/L/OD wet cells should be collected (~8 g from 1 L at 3 OD).<br/>5. After all the culture is harvested, resuspend the cell pellet in at least 5 mL of S30 buffer for each gram of wet cell weight.<br/>6. Centrifuge the cell suspension at 8,000 × g for 20–30 min.<br/>7. Discard the supernatant and freeze the washed cell pellet at −80°C ( see Note 12 ).

3.2. Biomass Processing

1. Break the frozen cell paste into small pieces and thaw in 1 mL of room temperature S30 buffer per gram cell paste ( see<br/>Note 13 ).<br/>2. Shake and stir the cell suspension periodically until it is well mixed. Once thawed, keep the cell suspension on ice if the lysis<br/>step cannot be done immediately. Rinse the homogenizer with S30 buffer before processing the cell suspension.

3. Lyse the cells with a single pass through an Avestin Emulsifl ex<br/>high pressure homogenizer at 17,500 psi ( see Note 14 ). The<br/>lysate should be cooled as quickly as possible after exiting the<br/>homogenizer, preferably through a cooling coil or heat<br/>exchanger. Keep the lysate on ice until it is all collected.

4. Centrifuge the lysate at 30,000 × g for 30 min at 4°C ( see<br/>Note 15 ).

5. Transfer the supernatant to a clean tube and repeat the<br/>centrifugation ( see Note 16 ).

6. Add 2 mL of pre-incubation mix for each 10 mL of clarifi ed<br/>lysate and place in a closed tube or bottle in a 37°C shaker with<br/>gentle shaking (~100 rpm) for 80 min ( see Notes 17 and 18 ).

<br/>7. Centrifuge the extract at 4,000 × g for 10 min.

8. Aliquot the supernatant into tubes and freeze in liquid<br/>nitrogen before storing at −80°C ( see Note 19 ).

3.3. Extract Testing<br/>Testing an extract consists of running a cell-free transcription/<br/>translation reaction to produce a model protein. The procedure<br/>is described below for the Cytomim system ( 5 ) ( see Note 20 )<br/>using chloramphenicol acetyl transferase (CAT) as a model protein.<br/>Ideally the model product would be similar to the candidate<br/>protein to be used in experiments. Modifi cations to the reaction<br/>conditions may be needed for disulfi de bond formation in the<br/>product ( 6– 8 ) .

The procedure below uses radiolabeling to quantify the product<br/>protein. There is also an enzymatic assay for CAT activity ( 9, 10 ) .<br/>Alternative model proteins can be used to enable other detection<br/>methods (i.e. fl uorescence for GFP production or luminescence for<br/>luciferase production).

The exact optimal expression conditions vary slightly for each<br/>batch of extract and potentially for each product, with magnesium<br/>concentration being the most sensitive. If the standard conditions<br/>do not provide acceptable activity, the magnesium concentration<br/>in the cell-free reaction should be varied about ±4 mM in 1 or<br/>2 mM increments to identify the optimum conditions. Optimizing<br/>plasmid, T7 RNA polymerase, potassium and extract concentrations<br/>can also help improve activity.

1. Thaw an aliquot of extract and all the cell-free reagents in<br/>Table 1 .

2. Mix reagents in the order listed in Table 1 , adding the plasmid<br/>and extract last ( see Note 21 ).

3. Incubate at 37°C for 3–5 h ( see Note 22 ).

4. While the reactions are running, cut two small slips of Whatman<br/>3MM filter paper for each reaction and label them with<br/>pencil. Pierce each strip with a straight pin and stick the pin<br/>in a sheet of Styrofoam covered in aluminum foil (shipping<br/>cooler lids work well). Make sure the fi lter paper is held above<br/>the foil by the pin so that the sample will not spread from<br/>the fi lter to the foil.

5. At the desired endpoint, for each reaction, spot 5 m L of<br/>cell-free reaction each on two separate fi lter paper slips.

6. Dry the slips for 15 min under an incandescent lamp or<br/>overnight on the bench.

7. Place one set of slips (with the pins still in them) in a small<br/>beaker and wash three times with cold 5% (w/v) trichloroacetic<br/>acid (TCA) for 10 min. Wash once with ethanol ( see Note 23 ),<br/>then return to the Styrofoam sheet. Dry under a lamp for&nbsp;<br/>15 min or overnight on the bench. Ensure that the washed<br/>slips can be differentiated from the unwashed slips.

8. Remove the pins from the fi lters and place each slip into a<br/>separate scintillation vial, add cocktail, and count in a liquid<br/>scintillation counter.

9. Calculate the protein produced with the following formula:<br/>[Product ](mg/L)= Counts (washed) / Counts (unwashed)<br/>x [Leucine ]x Y x X,

where [Leucine] is the total concentration of leucine (2 mM in<br/>this case), Y is the molecular mass of the product, and X is the<br/>number of leucine residues in the product

<br/>4. Notes

1. Other strains can be used (MRE600, BL21, etc.). A strain with<br/>reduced RNase activity (i.e. rna and/or rne mutations) may give<br/>better results. Make sure the growth medium contains any<br/>required nutritional supplements if an auxotrophic strain is used.<br/>with glacial acetic acid. The pH of the fi nal S30 buffer usually is<br/>not checked or adjusted. A large container of distilled water can<br/>be chilled and used for making cold S30 buffer as needed. If<br/>desired, the water can be sterilized prior to chilling.

2. Tunair flasks are available from IBI Scientific and several<br/>distributors. The fl asks have a higher oxygen transfer rate than<br/>standard baffl ed glass fl asks, so they support higher growth<br/>rates and cell densities. Alternatively, the culture volume can be<br/>split between multiple glass fl asks (typically no more than<br/>400 mL in a 2-L glass fl ask).

3. The specifi c medium used for the inoculum is not critical, but<br/>preferably it would be the same as the main culture medium.<br/>However, in my experience, 2YT works better for overnight<br/>inoculum cultures than 2YTPG. If 2YTPG is used for the inoculum,<br/>the lag phase may be longer than with a 2YT inoculum.

4. Other media such as LB, 2YT, or a defi ned medium can be<br/>used, but 2YTPG provides a good balance of cost, simplicity,<br/>and reasonable cell density (which affects the yield of extract).<br/>Also, 2YTPG will produce better extract than 2YT for longer<br/>cell-free reactions ( 2 ) . The 2YTPG medium should be sterile<br/>filtered instead of autoclaved to avoid carmelization of the glucose.<br/>Alternatively, a separate 50% (v/v) glucose stock can be<br/>made and added to the other medium components after autoclaving.<br/>The growth rate of the culture determines the ribosomal<br/>content of the extract ( 11 ) , so a medium which supports<br/>rapid growth ( m > 0.5 h −1 ) should be used for best results. If<br/>antifoam 204 is used, samples should be chilled to £ 25°C before<br/>measuring the OD to eliminate the cloudiness of the antifoam.<br/>Placing a 1 mL sample on ice for a couple minutes works well<br/>as long as the sample is mixed before OD measurement.

5. The optimum pH for E. coli is typically about 7.2–7.4, but can<br/>vary depending on the strain and medium used. Metabolic<br/>waste products generated from the glucose in the 2YTPG<br/>medium will cause the culture to become acidic as the cell<br/>density increases, so pH control can help sustain rapid growth<br/>to higher cell densities. If pH control is not possible, high<br/>quality extract can still be produced, but the culture may need<br/>to be harvested at a lower cell density. In addition to pH control,<br/>fed-batch fermentation methods can be used to reach<br/>higher cell densities (thus producing more extract per batch),<br/>but such methods are more complicated to set up and operate.<br/>There are many published strategies for fed-batch fermentations<br/>and a detailed description is beyond the scope of this<br/>chapter. If a fed-batch method is employed, it is important that<br/>it maintains a rapid growth rate to produce the highest ribosome<br/>content in the extract ( 11 ) .

6. S30 buffer is typically prepared as needed from purifi ed water<br/>and three separate 100× concentrates (1 M Tris acetate pH 8.2,<br/>1.4 M magnesium acetate, and 6 M potassium acetate) which<br/>are sterile fi ltered for increased stability and stored at room<br/>temperature. The pH of the 1 M Tris solution is adjusted to 8.2

7. It is convenient to prepare small amounts of the salt solution<br/>with varying magnesium concentrations for testing the magnesium<br/>optimum of each batch of extract. Typically, 8 mM magnesium<br/>in the cell-free reaction (equivalent to 80 mM in the<br/>10× salt solution) gives acceptable performance, but the optimum<br/>may vary from about 4 to 12 mM. All reagents for extract<br/>testing should be divided into small aliquots, frozen in liquid<br/>nitrogen, and stored at −80°C for the greatest stability.

8. Tyrosine is not soluble in the amino acid mixture and other<br/>amino acids may precipitate over time. This does not affect the<br/>performance as long as the mixture is thoroughly mixed immediately<br/>before use.

9. T7 RNA polymerase can be purchased from a number of suppliers.<br/>Kigawa recommended Ambion, Takara, and Promega<br/>products ( 12 ) . Alternatively, the polymerase can be prepared<br/>in-house as a His-tagged protein and purifi ed with a standard<br/>IMAC procedure ( 13 ) followed by dialysis or ultrafi ltration to<br/>remove the imidazole. As another option, Nevin and Pratt ( 14 )<br/>as well as Kim et al. ( 15 ) have expressed T7 RNA polymerase<br/>directly in the source cells used for extract preparation so that<br/>the extract already contains the T7 RNA polymerase.

10. Any vector designed for T7-driven expression should work in a<br/>cell-free system, but the specifi c vector design can affect productivity.<br/>Invitrogen sells vectors for cell-free expression that<br/>should be suitable for this system. Standard plasmid purifi cation<br/>kits produce suitable quality plasmid for cell-free reactions.

11. The maximum OD before stationary phase will depend on the<br/>strain, medium, and culture vessel used. Virtually any system<br/>should support at least 1 OD, so harvesting at this point will<br/>likely produce good extract (but less volume of extract). The<br/>A19 strain grown in 2YTPG in a Tunair fl ask with 280 rpm<br/>shaking can reach 5–8 OD in exponential phase if the pH is<br/>controlled. Without pH control, growth may start to slow<br/>around 3 OD as the pH drops. Usually, the culture will have<br/>a 1–2 h lag phase followed by 2–3 h of exponential growth.<br/>The density and age of the overnight culture will affect the lag<br/>time and duration of the growth phase. Checking the OD<br/>every 30 min once growth starts will provide good data for<br/>growth rate calculation and determination of the maximum<br/>OD with rapid growth.

12. It is most convenient to scrape the cell paste into a plastic<br/>sample bag (VWR #89004-424) and fl atten to a thin sheet<br/>before freezing. This takes up less freezer space than a centrifuge<br/>bottle and the frozen sheet can be broken up into smaller pieces<br/>to speed thawing. Also, if a large batch of cells is prepared, it can<br/>be split into portions for multiple extract preparations, for<br/>example, to test variations on the extract procedure. Frozen<br/>cells can be stored at −80°C for several months. If the rest of<br/>the extract preparation procedure will be done immediately,<br/>there is no need to freeze the cells.

13. Hanes et al. ( 4 ) used 4 mL of buffer per gram cell paste. Using<br/>more buffer will result in a more dilute cell lysate which will<br/>improve clarifi cation. However, it also results in a larger volume<br/>of cell suspension to be lysed and clarifi ed. Most likely, the<br/>buffer volume used does not have a strong impact on the total<br/>amount of extract activity produced. So the volume can be<br/>chosen to make processing most convenient for the lysis<br/>method used and the available equipment. The optimum volume<br/>of extract used for transcription/translation reactions will<br/>depend on the cell dilution factor. If more buffer is used to<br/>resuspend the cells, more extract will be needed in the transcription/<br/>translation reaction.

14. Avoid getting any air bubbles or foam in the homogenizer,<br/>otherwise the extract activity may be reduced. Other cell lysis<br/>methods (bead milling, French press, etc.) have been used for<br/>extract preparation. To my knowledge, there is no published<br/>study comparing the various lysis techniques, but Kigawa recommended<br/>glass bead milling and discouraged sonication ( 12 ) .

15. The centrifugation can be done at a lower g -force ( 15 ) . If bottles<br/>larger than 50 mL are used, extending the centrifugation<br/>time 10–20 min will help improve the clarifi cation.

16. The pellet material is very detrimental to extract performance,<br/>and the pellet is usually somewhat loose and fl uid. So, the supernatant<br/>should be transferred to a clean tube as soon as possible,<br/>preferably by using a pipette and leaving the bottom 10% or so<br/>on the pellet to avoid transferring any pellet material.

17. This pre-incubation step dissociates the polysomes and ribosomes<br/>in the extract into 30S and 50S ribosomal subunits ( 3 ) .<br/>Liu and co-workers showed that omitting the reagent additions<br/>to the pre-incubation did not affect bulk protein synthesis<br/>by the fi nished extract. Leaving out the additions simplifi es<br/>the process and greatly reduces the cost. However, without<br/>the reagent additions, the 70S ribosomes do not dissociate<br/>into subunits. Since some ribosome display translation reactions<br/>last only a few minutes, it may be important to have the ribosomes<br/>already dissociated in the extract for the best activity.<br/>Also, Hanes et al. ( 4 ) suggest better performance is achieved<br/>with a pre-incubation of 60 min at 25°C. In addition, their<br/>pre-incubation mix has slightly different concentrations of<br/>the components. A few experiments can determine the effects<br/>of these variations in any specifi c application.

18. Most extract preparation procedures include extensive dialysis<br/>after the pre-incubation. Typically this is done at 4°C for four<br/>45 min periods versus 20 volumes of S30 buffer (changed<br/>every 45 min) using 6–8,000 Da molecular weight cut-off<br/>dialysis tubing. The work of Liu et al. ( 3 ) showed little if any<br/>benefi t of the dialysis step for bulk transcription/translation<br/>reactions, so it was omitted from the procedure here.

19. Extract is usually stable for at least three freeze-thaw cycles.<br/>Frozen extract stored at −80°C is stable for at least a year. If a<br/>large batch of extract is made, it is convenient to make a few<br/>dozen single-use aliquots and then divide the rest of the extract<br/>into larger portions. When needed, a large aliquot can be<br/>thawed and split into another set of single-use aliquots. All<br/>extract samples should be frozen in liquid nitrogen and stored<br/>at −80°C. Slow freezing or storage at higher temperatures can<br/>reduce the extract activity.

20. Other energy generation systems have been developed for use<br/>in cell-free reactions. For more information, see the review by<br/>Calhoun and Swartz ( 16 ) . The Cytomim system requires<br/>inverted membrane vesicles to perform oxidative phosphorylation.<br/>These vesicles are formed during the high pressure<br/>homogenization lysis step and remain in the fi nal extract as<br/>prepared with this procedure. If a different cell lysis method is<br/>used, it may be necessary to use a different energy generation<br/>system in the cell-free reaction to achieve good performance.

21. If only a few reactions are planned, the recipe should be multiplied<br/>to ensure all the required volumes are reasonable to pipette<br/>accurately. The standard 15 m L reactions work well in 1.5-mL<br/>microcentrifuge tubes. If larger reactions are required, the reaction<br/>vessel may need to be changed. Voloshin and Swartz have<br/>investigated the effects of volume and reactor geometry ( 17 ) .

22. Some proteins express better at lower temperatures. If desired,<br/>a range of temperatures can be tested for each new product to<br/>find the optimum (typically 30°C–37°C). Protein synthesis<br/>may continue for 5 h or more ( 5 ) , so a time course should be<br/>run if maximum product titer is important.

23. The ethanol wash speeds up the drying process. It usually does<br/>not affect the results if it is omitted.

Recipe for a 15 μL cell-free transcription-translation reaction<br/>Reagent Stock concentration Reaction concentration Volume ( μL)<br/>Milli-Q water 5.5525<br/>10× salt solution 10× 1× 1.5<br/>10× master mix 10× 1× 1.5<br/>AA mix 50 mM each 2 mM 0.6<br/>Pyruvate 1 M 33 mM 0.5<br/>Oxalate 1 M 4 mM 0.06<br/>Putrescine 200 mM 1 mM 0.075<br/>Spermidine 200 mM 1.5 mM 0.1125<br/>CoA 20 mM 0.27 mM 0.2<br/>NAD 100 mM 0.33 mM 0.05<br/>14 C Leu 0.1 mCi/mL 1.7 μCi/mL 0.25<br/>T7 RNA polymerase 0.8 mg/mL 33 μg/mL 0.6<br/>Plasmid 0.5 mg/mL 13.3 μg/mL 0.4<br/>Extract 24% (v/v) 3.6