Editing S30 E. coli extract

== 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

== Protocol 2 ==

https://www.nature.com/articles/srep08663.pdf

'''Materials.''' E. coli BL21 StarTM (DE3) strain was purchased from Life Technologies (Grand Island, NY). Strain C495 (also reported as MCJ.495) is a genomically engineered variant of K12 MG1655 described previously48. E. coli total tRNA mixture (from strain MRE600) was purchased from Roche Applied Science (Indianapolis, IN). ATP, GTP, CTP, UTP, Phosphoenolpyruvate, 20 amino acids and other materials were purchased from Sigma (St. Louis, MO) without further purification. sfGFP was cloned into pY71 vector using NdeI and SalI restriction site and used as model protein in this study. T7 RNA Polymerase was obtained by affinity tag purification56.

'''Preparation of cell extracts'''<br/>E. coli BL21 Star™ (DE3) and C495 cells were grown in 2 × YTPG media at 34°C. We grew the strains at 34°C such that we could compare the strains. The MG1655-based C495 strain harbors a temperature sensitive λ-Red recombinase that requires growth at a lower temperature. Antibiotics were not used during cell growth. The cells were grown in a BIOSTAT C-plus fermentor (Sartorious AG, Goettingen, Germany) at 600 rpm for 10 L large-scale cultures. For preparing 1 L scale cultures, cells were grown in 2.5 L baffled tunair shake flasks (IBI Scientific, Peosta, IA) in a 34°C incubator with vigorous shaking at 250 rpm. For smaller scale cultivation (50 to 100 mL culture), 300 mL baffled tunair flasks were used. Unless otherwise specified in the text, the cultured cells were monitored by spectrophotometry (Genesys 10S UV-Vis, Thermo Fisher Scientific, Waltham, MA) until OD600 reached at 3.0. The maximum OD600 for BL21 Star™ (DE3) and C495 strains measured after 22 h culture that reached saturation were ~10 and ~8, respectively. The cells were harvested in the middle of exponential growth phase by centrifuging at 5000 RCF at 4°C for 15 min and were washed three times with cold Buffer A. Buffer A contained 10 mM Tris-acetate (pH 8.2), 14 mM magnesium acetate, 60 mM potassium glutamate, and 2 mM dithiothreitol. After final wash and centrifugation, the pelleted wet cells were weighed, flash frozen in liquid nitrogen, and stored at −80°C. The thawed cells were suspended in 1 mL of Buffer A per 1 g of wet cell mass. For obtaining standard “control” large-scale cultured cell extract, suspended cells were disrupted by EmulsiFlex-C3 homogenizer (Avestin, Ottawa, Canada) with single pass at a variable pressure of 20,000 to 25,000 psig. In order to remove cell debris and insoluble components in the cell lysate, the lysate was centrifuged twice at 30,000 RCF at 4°C for 30 min. The supernatant was then incubated at 37°C for 60 min with gentle shaking (250 rpm) and centrifuged at 15,000 RCF at 4°C for 15 min. In order to lyse cells by sonication, thawed and suspended cells were transferred into 1.5 mL microtube and placed in an ice-water bath to minimize heat damage during sonication. The cells were lysed using a Q125 Sonicator (Qsonica, Newtown, CT) with 3.175 mm diameter probe at frequency of 20 kHz and 50% of amplitude. The input energy (Joules) was monitored and recorded during sonication. The lysate was then centrifuged once at 12,000 RCF at 4°C for 10 min. The run-off reaction (37°C at 250 rpm) and second centrifugation (10,000 RCF at 4°C for 10 min) were performed for strain C495. However, in the case of strain BL21 Star™ (DE3), the run-off reaction and second centrifugation step were not needed. The total amount of protein in cell extract was quantified by Bradford assay. All extracts contained 40.7 ± 2.6 mg/mL of total E. coli proteins. All of prepared cell extract was flash frozen in liquid nitrogen and stored at −80°C until use.

'''Cell-free protein synthesis'''<br/>The CFPS reactions were carried out in a 1.5 mL microtube in the incubator. The standard reaction mixture for CFPS consists of the following components in a final volume of 15 μL: 1.2 mM ATP; 0.85 mM each of GTP, UTP, and CTP; 34.0 μg mL−1 L-5-formyl-5, 6, 7, 8-tetrahydrofolic acid (folinic acid); 170.0 μg mL−1 of E. coli tRNA mixture; 130 mM potassium glutamate; 10 mM ammonium glutamate; 12 mM magnesium glutamate; 2 mM each of 20 amino acids; 10 μM of L-[14C(U)]-leucine (11.1 GBq mmol−1, PerkinElmer, Waltham, MA); 0.33 mM nicotinamide adenine dinucleotide (NAD); 0.27 mM coenzyme-A (CoA); 1.5 mM spermidine; 1 mM putrescine; 4 mM sodium oxalate; 33 mM phosphoenolpyruvate (PEP); 13.3 μg mL−1 plasmid; 100 μg mL−1 T7 RNA polymerase, and 27% v/v of cell extract. The CFPS reactions were carried out at 37°C for 4 hours.



== Protocol 3 ==

[http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0165137 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0165137]<br/>Preparation of S30-T7 lysate

1.1. Streak the bacteria (transformed with pAR1219) on an LB-agar plate.

AR1219: expressing T7 RNA Polymerase&nbsp;&nbsp;[https://www.sigmaaldrich.com/catalog/product/sigma/t2076?lang=en&region=US https://www.sigmaaldrich.com/catalog/product/sigma/t2076?lang=en&region=US]

1780:&nbsp;pT7A3-T7-RNAPol-Y639F, 0.5 mM IPTG

1080 pBad24-T7-RNAPol:&nbsp;0.2% arabinose

1.2. Use a single colony to inoculate 10 ml LB media in 100 mL flask, and grow it overnight at 37°C with shaking at 250 rpm on a floor incubator shaker. This step will obtain a starter solution (duplicate).

1.3. Inoculate each one of the 500 ml TB media inside a 2 liters flask using the 10 ml starters.

1.4. Grow the culture at 37°C until it reaches OD600, ~= 1, by monitoring the culture's OD at 600 nm using a spectrophotometer. Once the required OD is achieved, add 0.4 mM IPTG.

1.5. Grow the culture at 37°C until it reaches OD600, ~= 4.

1.6. Centrifuge at 7,000 x g for 10 min at 4°C.

1.7. Re-suspend each pellet in 500 ml of S30 lysate buffer, and centrifuge at 7,000 x g for 10 min at 4°C.

S30 lysate buffer:10 mM Tris-Acetate, pH 7.4, 10 mM Magnesium acetate, 60 mM potassium acetate, 1 mM DTT

'''1 L of 100x stock'''

1M Tris-Acetate pH 7.2, 121.14 g Trizma Base (Sigma T6066) Adjust pH to 7.2 with Glacial Acetic Acid

1.4 M Mg(Acetate)2,&nbsp; 300.16 g Mg(OAc)2 Tetrahydrate (Sigma 228648)

6.0 M K(Acetate),&nbsp; 588.90 g K(OAc) (Mallinckrodt 6700)

'''10X S30 buffer:'''

100 mM Tris acetate, pH 8.2

160 mM potassium acetate

140 mM magnesium acetate

pH 8.3 with potassium hydroxide

'''&nbsp;S30 buffer A''': 1X S30 buffer containing 0.5 mM PMSF, 1 mM DTT and 7.2 mM -mercaptoethanol

1.8. Resuspend in 15 ml of S30 lysate buffer.

1.9. Break the cells by one pass through a high pressure homogenizer at a working pressure of 15,000 psi, with an air pressure of 4 bar.

1.10. Add 100 pL of 0.1 M DTT to each 10 ml of homogenized suspension.

1.11. Centrifuged the suspension at 24,700 x g or 13,000 x g for 30 min at 4°C.

1.12. Divide the supernatant into aliquots of 200 pL into 1.5 ml tubes.

1.13. Freeze the tubes immediately by liquid nitrogen and store at -80°C for further use.&nbsp;

A single colony was used to inoculate fresh LB media, and was grown overnight at 37°C with orbital shaking of 250 rpm. This was used as a starter to inoculate fresh Terrific Broth (TB) the following day at a 1:50 starter:medium ratio. The media were sterilized and supplemented with ampicillin at 50 μg/ml. The culture was grown at 37°C to OD&lt;/span&gt;<span style="box-sizing: border-box; vertical-align: 0px; position: relative; top: 0.5ex; font-family: arial;">600</span><span style="font-family: arial;">≈1, upon which 0.4 mM 0.2 μm filtered Isopropyl β-D-1-thiogalactopyranoside (IPTG) solution was added. The culture was further grown at 37°C until it reached OD</span><span style="box-sizing: border-box; vertical-align: 0px; position: relative; top: 0.5ex; font-family: arial;">600</span><span style="font-family: arial;">≈4 and was centrifuged at 7,000 x g for 10 min at 4°C. The pellet was re-suspended in the same volume (1:1 v/v) of cold S30 lysate buffer containing: 10mM Tris-acetate at pH = 7.4, 14mM magnesium acetate, 60 mM potassium acetate, 1 mM 0.2 μm filtered dithiothreitol (DTT) and 0.5 ml/L 2-mercaptoethanol. Subsequently, the suspension was centrifuged again and was resuspended in 15 ml of S30 lysate buffer. Then, the cells were broken by one pass through an emulsiFlex-C3 high pressure homogenizer (Avestin, Mannheim, Germany) that was pre-cooled to 4°C and at a working pressure of 15,000 psi, with an air pressure of 4 bar. One-hundred μl of 0.1 M DTT was added to each 10 ml of homogenized suspension. Finally, the suspension was centrifuged at 24,700 x g or 13,000 x g for 30 min at 4°C divided into aliquots of 200 μl, frozen by liquid nitrogen and stored at -80°C for further use.</span>

<br/><span style="font-family: arial;">A detailed protocol for the preparation of the S30-T7 lysate is described in Table 1. Briefly, S30-T7 lysates were prepared from E. coli BL21(DE3) and MRE600 transformed with TargeTron® vector pAR1219 (Sigma-Aldrich, Rehovot, Israel). Bacteria glycerol stocks were streaked on an agar Luria Bertani (LB) plate. A single colony was used to inoculate fresh LB media, and was grown overnight at 37°C with orbital shaking of 250 rpm. This was used as a starter to inoculate fresh Terrific Broth (TB) the following day at a 1:50 starter:medium ratio. The media were sterilized and supplemented with ampicillin at 50 μg/ml. The culture was grown at 37°C to OD600≈1, upon which 0.4 mM 0.2 μm filtered Isopropyl β-D-1-thiogalactopyranoside (IPTG) solution was added. The culture was further grown at 37°C until it reached OD600≈4 and was centrifuged at 7,000 x g for 10 min at 4°C. The pellet was re-suspended in the same volume (1:1 v/v) of cold S30 lysate buffer containing: 10mM Tris-acetate at pH = 7.4, 14mM magnesium acetate, 60 mM potassium acetate, 1 mM 0.2 μm filtered dithiothreitol (DTT) and 0.5 ml/L 2-mercaptoethanol. Subsequently, the suspension was centrifuged again and was resuspended in 15 ml of S30 lysate buffer. Then, the cells were broken by one pass through an emulsiFlex-C3 high pressure homogenizer (Avestin, Mannheim, Germany) that was pre-cooled to 4°C and at a working pressure of 15,000 psi, with an air pressure of 4 bar. One-hundred μl of 0.1 M DTT was added to each 10 ml of homogenized suspension. Finally, the suspension was centrifuged at 24,700 x g or 13,000 x g for 30 min at 4°C divided into aliquots of 200 μl, frozen by liquid nitrogen and stored at -80°C for further use.</span>



{|
|-
| <span style="font-family: arial;">Cell-free reaction component</span>
| <span style="font-family: arial;">Final concentration in solution&nbsp;</span>
|-
| <span style="font-family: arial;">HEPES-KOH (pH = 8)</span>
| <span style="font-family: arial;">55 mM&nbsp;</span>
|-
| <span style="font-family: arial;">Magnesium acetate</span>
| <span style="font-family: arial;">14 mM&nbsp;</span>
|-
| <span style="font-family: arial;">Potassium acetate</span>
| <span style="font-family: arial;">50 mM&nbsp;</span>
|-
| <span style="font-family: arial;">Ammonium acetate</span>
| <span style="font-family: arial;">155 mM&nbsp;</span>
|-
| <span style="font-family: arial;">Polyethylene glycol (PEG)</span>
| <span style="font-family: arial;">3% (v/v)&nbsp;</span>
|-
| <span style="font-family: arial;">3-phosphoglycerate (3-PGA)</span>
| <span style="font-family: arial;">40 mM&nbsp;</span>
|-
| <span style="font-family: arial;">20 amino acids</span>
| <span style="font-family: arial;">2.5 mM&nbsp;</span>
|-
| <span style="font-family: arial;">Adenine triphosphate (ATP)</span>
| <span style="font-family: arial;">1.2 mM&nbsp;</span>
|-
| <span style="font-family: arial;">Guanidine triphosphate (GTP)</span>
| <span style="font-family: arial;">1 mM&nbsp;</span>
|-
| <span style="font-family: arial;">Uridine triphosphate (UTP)</span>
| <span style="font-family: arial;">0.8 mM&nbsp;</span>
|-
| <span style="font-family: arial;">S30-T7 lysate</span>
| <span style="font-family: arial;">30% (v/v)&nbsp;</span>
|-
| <span style="font-family: arial;">DNA template</span>
| <span style="font-family: arial;">10 pg/mL&nbsp;</span>
|-
| colspan="2" | <span style="font-family: arial;">DNase, RNase free H2O to reaction volume&nbsp;</span>
|}



<span style="font-family: arial;">A. Materials and solutions required:&nbsp;</span>

<span style="font-family: arial;">1M HEPES-KOH (pH = 8)</span>

<span style="font-family: arial;">1M magnesium acetate</span>

<span style="font-family: arial;">1M potassium acetate</span>

<span style="font-family: arial;">5.2 M ammonium acetate</span>

<span style="font-family: arial;">50% (v/v) Polyethylene glycol 6000 (PEG)</span>

<span style="font-family: arial;">0.5 M 3-phosphoglycerate (3-PGA)</span>

<span style="font-family: arial;">Notes:&nbsp;</span>

<span style="font-family: arial;">50 mM of 17 amino acids These 17 amino acids are alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, and valine. 50 mM of 3 amino acids These 3 amino acids are tryptophan, phenylalanine, and tyrosine.</span>

<span style="font-family: arial;">100 mM Adenine triphosphate (ATP)</span>

<span style="font-family: arial;">50 mM Guanidine triphosphate (GTP)</span>

<span style="font-family: arial;">100 mM Uridine triphosphate (UTP)</span>

<span style="font-family: arial;">S30-T7 lysate Prepared according to Table 1</span>

<span style="font-family: arial;">DNase, RNase free H2O B. Equipment:</span>

<span style="font-family: arial;">Notes: Floor incubator shaker or a Thermomixer® C. Procedure:&nbsp;</span>

== <span style="font-family: arial;">Protocol 4</span> ==

[http://ajournal.co.uk/pdfs/volume1/Vol.1%20(1)%20Article%205.pdf http://ajournal.co.uk/pdfs/volume1/Vol.1%20(1)%20Article%205.pdf]

Cell-free protein synthesis for analysis by NMR spectroscopy,&nbsp;British Journal of Science 45 September 2011, Vol. 1 (1)

'''3.1.1. Day One: Buffer preparation and overnight culture&nbsp;'''

1. Prepare and autoclave stock solutions of glucose (2 M, 224 mL), thiamine (1 mg/mL, 200 mL), 10X S30 buffer (2 L) and 50% PEG 8000 (2 L) as listed in the materials section. Stir the mixture of 50% PEG 8000 at room temperature overnight. Autoclave the stock solutions of glucose, thiamine and 10X S30 buffer, 1 L of LBT medium and 20 L of MQ water for diluting the 10X S30 buffer stock. Autoclave a large glass funnel and two 250 mL measuring cylinders required for measuring the glucose and thiamine solutions to be added to the medium on day three.

&nbsp;LBT: 10 g/L tryptone, 5 g/L yeast extract, 5 g/L sodium chloride, 2.5 mL/L 1 M sodium hydroxide, 0.5 mg/L thymine

2. Grow a 10 mL culture of '''BL21 DE3 star''' cells (invitrogen) in LBT overnight at 37 oC. If the BL21 DE3 Rosetta pRARE strain is used, include 33 ug/mL of chloramphenicol.

3. After autoclaving, store MQ water and 10X S30 buffer at 4 oC.

'''3.1.2. Day Two: Z-medium preparation and overnight 1 L culture'''

1. Prepare 5 L of 4X Z-medium buffer.

2. Add 5 L of Z-medium concentrate, 4.5 mL of antifoam and 14 L of MQ water into the fermenter. This gives a total volume of 19 L. When 1 L of overnight culture is added to this the following day, 20 L of 1X Zmedium will result.

4X Z-medium: 165 mM potassium dihydrogen phosphate, 664 mM potassium phosphate dibasic, 40 g/L yeast extract

3. Sterilise the medium at 121 oC using the sterilisation function of the fermenter.

4. Calibrate the pO2 electrode of the fermenter to 0% after 25 min at 121 oC.

'''3.1.3. Day Three: 20 L culture and processing of cell pellets'''

1. Equilibrate the fermenter at 37 oC, with 100% air and mixing rate at 395 rpm.

2. Prior to addition of the overnight culture, remove a sample from the outlet at the base as a blank for later measurement of the absorbance of the culture at 595 nm (OD).

3. Add the 1 L overnight culture, 224 mL of 2 M glucose and 200 mL of 1 mg/mL thiamine to the Z-medium with sterile glass funnel. .

4. Allow these to mix before removing a time zero sample for OD measurement.

5. Remove samples for OD measurements every 30 minutes until the culture reaches an OD of 3. This density should be achieved after 3-4 hours.

6. Fill a large sink or tub with a large amount of crushed ice in preparation for the next steps.

7. Drain the culture into 5 L conical flasks and immediately place them on ice. Weigh empty 1 L tubes.

8. Harvest the cells by centrifugation at 4 oC for 12 min at 10,000 g in 1 L tubes. Use pre-weighed tubes so that the pellet mass can be determined later.

9. Resuspend the cell pellets in 400 mL of 1X S30 buffer A. Centrifuge the resuspended cells for 10 min at 10,000 g and 4 oC.

10. Remove the supernatant and pack the pellet by centrifugation for a further 5 minutes. Remove the remaining supernatant with a pipette.

11. Snap freeze the pellets in liquid nitrogen, then store them at –80 oC

'''3.1.4. Day Four: Preparation of the S30 extract''' 1. Remove the pellets from the freezer and defrost slowly on ice for at least one hour prior to resuspension.

2. Resuspend the pellets in 400 mL of S30 buffer A, and centrifuge for 12 min at 10,000 g.



4. Resuspend the pellets in 1.3 mL of S30 buffer A&nbsp;per gram of cells.

5. Subject the suspension to a single pass in a French press at a pressure of 6,000 psi. If the lysate is very viscous pass it through the French press again.

6. Centrifuge the lysed cells at 5,200 g at 4 oC for 15 min to remove any cell debris in 1 L tubes. Transfer the supernatant to clean tubes.

7. Centrifuge the supernatant at 30,000 g at 4 oC for 1 h to produce the S30 fraction.

8. Prepare 3 batches of 3 L each of S30 buffer B. Dialyse the S30 fraction in Spectrapor # 4 dialysis tubing against each 3 L batch of S30 buffer B for one hour at 4 oC, i.e. for three hours in total.

S30 buffer B: 1X S30 buffer containing 1 mM DTT

8a. Optional: Take 1/3 of S30 extract before the following concentration step. Snap-freeze and store it at -80 oC.

9. Transfer the dialysis tubes containing the S30 fraction to a large evaporating dish and dialyse against two batches of 1L of 50% PEG 8000. Place the dish on a shaking tray to speed dialysis, and leave for 2 hours at 4 oC.

10. Change the 50% PEG buffer and repeat until the volume of the extract has been reduced to approximately half volume of the S30 extract (about 2h). It is measured by visual comparison with dialysis tubing containing 60 mL of water.

11. The concentrated S30 fraction is then dialysed against a further 3 L of 1X S30 buffer B for 15 minutes to remove the residual PEG 8000 buffer.

12. Measure the volume of the extract. Centrifuge the S30 extract if the level of precipitation is high.

13. Dispense the extract into 1 mL aliquots in 1.5 mL Eppendorf tubes.

14. Snap-freeze the aliquots in liquid nitrogen and store at -80 oC.

12a. Place the dialysis bag containing the S30 extract into a further 2 L of S30 buffer C for dialysis overnight at 4 oC.

13a. Place the dialysis bag containing the S30 extract into a 250 mL Pyrex glass bottle, filled with the S30 buffer C from the overnight dialysis. Prewarm that S30 buffer C to 42 oC.

For each new batch of S30 extract, the concentrations of magnesium acetate and the extract itself must be optimized. A series of reactions using between 9-25 L S30 extract per 100 L reaction mixture and between 15-25 mM magnesium acetate should be sufficient to find the optimal concentrations of each.

{|
|-
| Reagent&nbsp;
| Stock conc.
| MW
| Dry weight (g)<br/>
| Stock volume (mL)<br/>
| Final conc. in reaction mixture<br/>
| Supplier<br/>
| Storage (oC)<br/>
|-
| HEPES
| 2.0 M pH 7.5
| 238.3
| 95.35
| 200
| 55 mM
| Sigma
| RT
|-
| DTT
| 0.5 M
| 154.25
| 3.85
| 50
| 1.7 mM
| Sigma
| –20
|-
| ATP
| 96 mM
| 551.14
| 0.529
| 10
| 1.2 mM
| Sigma
| –80
|-
| rNTP
| 25 mM each
| 527.1
| 0.132
| &nbsp;
| 0.8 mM
| Sigma
| &nbsp;
|-
| &nbsp;
| &nbsp;
| 523.18
| 0.131
| &nbsp;
| 0.8 mM
| Sigma
| &nbsp;
|-
| &nbsp;
| &nbsp;
| 484.14
| 0.121
| &nbsp;
| 0.8 mM
| Sigma
| &nbsp;
|-
| Total
| &nbsp;
| &nbsp;
| &nbsp;
| 10
| 0.8 mM
| &nbsp;
| –80
|-
| Cyclic AMP
| 100 mM
| 351.2
| 0.351
| 10
| 0.64 mM
| Sigma
| –80
|-
| Folinic Acid&nbsp;
| 10 mM
| 511
| 0.0511
| 10
| 68 µM
| Sigma
| –80
|-
| Ammonium acetate (monohydrate)
| 9.2M
| 77.08
| 35.5
| 50
| 27.5 mM
| Ajax Finechem
| –80
|-
| tRNA
| 17.5 mg/mL
| &nbsp;
| 0.0175
| 1
| 0.175 mg/mL
| Roche
| –20
|-
| Creatine kinase
| 10 mg/mL
| 18 kDa
| 0.1
| 10
| 250 µg/mL
| Roche
| –80
|-
| colspan="2" | T7 RNA polymerase
| &nbsp;
| &nbsp;
| &nbsp;
| 93 µg/mL
| &nbsp;
| –20
|-
| pKO1166
| &nbsp;
| &nbsp;
| &nbsp;
| &nbsp;
| 32 µg/mL
| &nbsp;
| –20
|-
| Plasmid
| &nbsp;
| &nbsp;
| &nbsp;
| &nbsp;
| 16 µg/mL
| &nbsp;
| –20
|-
| S30 extract
| &nbsp;
| &nbsp;
| &nbsp;
| &nbsp;
| &nbsp;~225 µL/mL
| &nbsp;
| –80
|-
| Potasisum glutamate
| 4 M&nbsp;
| 203.23
| 40.65
| 50
| 208 mM&nbsp;
| Sigma
| –80
|-
| Magnesium acetate
| 1.07 M&nbsp;
| 214.45
| 11.47
| 50
| ~19.3 M
| Ajax Finechem
| RT
|-
| Creatine phosphate
| 1 M
| 327.14
| 16.36
| 50
| 80 mM
| Sigma
| –80
|-
| L-Alanine
| 100 mM
| 89.09
| 0.089
| 10
| 2 mM
| Sigma
| –80
|}





3.3. Preparation of the amino-acid stock solutions

The amino-acid stock solutions are prepared in three groups, water-soluble, acid-soluble, and base-soluble. Each amino acid is present in the stock solutions at a 50 mM concentration. The acid-soluble and basesoluble combinations are dissolved in 1 M hydrochloric acid and 1 M potassium hydroxide, respectively, such that when they are mixed, the pH of the solutions is close to neutral. The stocks can be kept on ice but the complete mixture must be mixed at room temperature to avoid precipitation at the final near-neutral pH. For preparations with individual amino acids provided in isotope labelled form, the stocks are prepared minus the amino acid of interest. The labelled amino acid is added later to the final reaction mixture from a stock of a concentration adjusted for economical usage of the labelled amino acids (14). The concentrations of the individual 100X 15N stock solutions are also listed in Table 2.&nbsp;

{|
|-
| colspan="3" | Water soluble mixture - dissolved in 10 mL water
|-
| Amino acid
| Weight (mg)
| 15N stock (mM)
|-
| Ala
| 44.5
| 100
|-
| Arg
| 105
| 35
|-
| Gly
| 37.5
| 100
|-
| His
| 105
| 15
|-
| Lys
| 91.3
| 100
|-
| Pro
| 57.6
| n/a
|-
| Ser
| 52.6
| 100
|-
| Thr
| 59.6
| 15
|-
| Val
| 58.6
| 100
|-
| colspan="3" | Acid soluble mixture - dissolved in 10 mL 1 M HCl
|-
| Amino acid
| Weight (mg)
| 15N stock (mM)
|-
| Asn
| 66
| 35
|-
| Asp
| 66.5
| 100
|-
| Cys
| 60.1
| 35
|-
| Glu
| 73.5
| 100
|-
| Gln
| 73.1
| 100
|-
| Leu
| 65.6
| 100
|-
| Met
| 74.6
| 100
|-
| Trp
| 102
| 5
|-
| Tyr
| 90.6
| 5
|-
| colspan="3" | Base soluble mixture - dissolved in 10 mL 1 M KOH
|-
| Amino acid
| Weight (mg)
| 15N stock (mM)
|-
| Ile
| 65.5
| 15
|-
| Phe
| 82.5
| 35
|}

To same procedure is used to prepare each of the three types of amino acid stock solutions.

1. Weigh out the individual amino acids in the quantities listed in Table 2.

2. Place all of the amino acids into a 10 mL tube.

3. Add 10 mL of the appropriate solvent to the mixture and vortex until dissolved. 4. Store the amino-acid stock at -80 oC.



3.4. Carrying out the cell-free reaction

3.4.1. Preparation of the reaction chamber

1. Soak one 10 cm length of Spectrapor #2 dialysis tubing per reaction in MQ water for 5 min, then tie a knot as close as possible to one end. Trim the knot to have minimal overhang.

2. Using clean scissors, prepare one 500 L Eppendorf tubes per reaction by cutting off the lid and hinge sections. The lid is retained and the hinge is discarded. Removal of the hinge allows the tube to fit inside a 10 mL tube. The bottom 0.5 cm of the tube is then sliced off using a scalpel.

3. Insert each cut Eppendorf tube into the top of a pre-tied length of dialysis tubing, as shown in Fig. 1. The Eppendorf tube ensures the contents of the dialysis tube are readily accessible. Remove the dialysis tubing from the MQ water and insert the Eppendorf tube immediately prior to use, so that the tubing does not dry out before assembly of the complete reaction.&nbsp;

Fig. 1: Schematic drawing of the assembly of the cut Eppendorf tube in the knotted dialysis bag to provide a readily accessible container for the reaction mixture.

'''3.4.2. Preparation of the reaction mixtures'''



Table 3: 10X B solution for a 500 µL reaction with 5 mL outside buffer

{| border="0" cellspacing="0" cellpadding="0"
|-
| colspan="2" | Stock solution µL
|-
| rNTP
| 179.5
|-
| HEPES
| 162.5
|-
| ATP
| 70
|-
| Water
| 38.5
|-
| Folinic acid
| 38
|-
| Cyclic AMP
| 35
|-
| DTT
| 19
|-
| Ammonium acetate
| 17
|-
| Total
| 559.5
|}



{| border="0" cellspacing="0" cellpadding="0"
|-
| colspan="3" | Table 4: Components of a 500 µL reaction mixture and 5 mL outside buffer
|-
| colspan="3" | Stock solution Reaction mixture (µL) Outside
|-
| buffer<br/>
| (µL)
| 
|-
| 10X B
| 50
| 500
|-
| Creatine phosphate
| 40
| 400
|-
| Potassium glutamate
| 26
| 260
|-
| Magnesium acetate
| 7
| 90
|-
| L-Alanine
| 5
| 50
|-
| Amino acid mixture
| 35
| 335
|-
| Creatine kinase
| 12.5
| 500
|-
| T7 polymerase vector
| 8
| 500
|-
| S30 extract
| 112.5
| 500
|-
| Vector of interest
| 8
| 500
|-
| tRNA
| 5
| 500
|-
| Water (adjust)
| 190
| 3365
|-
| total
| 500
| 5000
|}

3.4.2.1. Mixing the amino-acid stock solutions for use in a 500 µL reaction mixture with 5 mL outside buffer

1. Pipette 37.5 µL water into a 1.5 mL Eppendorf tube.

2. Add 112.5 µL water soluble stock and pipette up and down to mix well.

3. Add 112.5 µL acid soluble stock and pipette up and down to mix well.

4. Add 112.5 µL base soluble stock and pipette up and down to mix well.&nbsp;

3.4.2.2. Preparation of the 10X B mixture

Working on ice, add the stock solutions (described in Table 1) in the quantities listed in Table 3 in a 1.5 mL Eppendorf tube.

3.4.2.3. Preparation of the reaction mixture and outside buffer

Working on ice, mix the components listed in Table 4 for the reaction mixture in a 1.5 mL Eppendorf tube. Leave the reaction mixture on ice whilst the outside buffer is prepared in a 10 mL tube. Adjust the pH of the outside buffer using 1 M KOH prior to making up the final volume with water.





The quantities listed in this section are for one 500 L reaction mixture with 5 mL outside buffer. An Excel spreadsheet is available online ([http://rsc.anu.edu.au/~go http://rsc.anu.edu.au/~go]) that facilitates scaling to different volumes, number of reactions or different vector concentrations. The quantities of vectors listed assume stock solutions with a concentration of 1 mg plasmid per mL to achieve final concentrations in the reaction mixture of 16 g/mL, as per Table 1. If different volumes of vector solutions are to be added, adjust the quantity of water so that the final volume of the reaction is 500 L. The pH of the outside buffer must be adjusted to 7.5 using 1 M KOH, and the volume of water added adjusted accordingly. T7 RNA polymerase must be supplied if the expression of the target gene is under control of the T7 promoter. The pKO1166 vector is used in this example rather than purified T7 RNA polymerase so that fresh polymerase is continuously generated during the reaction (22). In most cases, pKO1166 delivers higher protein yields than purified T7 RNA polymerase, whereas experiments with incorporation of non-natural amino acids may depend on provision of the purified polymerase, which should be supplied at a final concentration of 32 g/mL. When using the purified polymerase enzyme there is no need for additional Lalanine to be added to the reaction mixture. The water volume to be added must be adjusted accordingly (21). The synthesis and purification of T7 RNA polymerase from a strain containing pKO1166 has been described in (14).





== PROTOCOL 5 ==

For small-scale in vitro synthesis of unlabeled proteins, the inner-chamber reaction mixtures (total volume 100 ll) contained 55 mM HEPES/ KOH (pH 7.5), 1.7 mM dithiothreitol, 1.2 mM ATP, 0.8 mM each of CTP, GTP and UTP, 0.64 mM 3¢,5¢-cyclic AMP, 68 lM folinic acid, 27.5 mM ammonium acetate, 208 mM potassium glutamate, 80 mM creatine phosphate, 25 lg creatine kinase, 1 mM of each of the twenty L-amino acids, 15 mM magnesium acetate, 17.5 lg E. coli total tRNA, 18 ll of Rosetta (kDE3)/pRARE S30K extract, 1.6 lg of supercoiled plasmid DNA, 150 units of RNase inhibitor, and either 9.3 lg of purified T7 RNAP or 3.2 lg of supercoiled plasmid pKO1166. Since T7 RNAP contains 100 alanine residues, the concentration of L-Ala was doubled when using pKO1166. Cell-free protein synthesis was carried out in Spectrapor 2 dialysis tubing with a nominal size cutoff of 12–14 kDa for 8 h at 37 C with gentle shaking in 3 ml of outer chamber solution. The inner chamber assembly and the outer chamber buffer were housed within a screw-capped 10 ml polypropylene tube. The outer chamber solution had the same composition as the inner chamber reaction mixture, except that S30K extract, tRNA, plasmid DNA, pKO1166 or T7 RNAP, creatine kinase and RNase inhibitor were omitted, and the concentration of magnesium acetate was increased to 19.3 mM. After incubation, the soluble and insoluble components of the inner chamber were separated by centrifugation (30,000 · g, 1 h at 4 C)

== Protocol 6 ==



https://www.researchgate.net/publication/259270251_Synthesis_of_23_mgmL_of_Protein_with_an_all_E_coli_Cell-Free_Transcription-Translation_System

The crude extract was prepared as above<br/>using the BL21 Rosetta2 strain and with the following slight modifications.<br/>S30A buffer composition: 14 mM Mg-glutamate, 60 mM<br/>K-glutamate, 50 mM Tris buffered with acetic acid to pH 8.2. S30B<br/>buffer composition: 14 mM Mg-glutamate, 150 mM K-glutamate<br/>buffered to pH 8.2 with Tris. The lysate was prepared with a cell<br/>press at a pressure of 16,000 LB.<br/>2.2. Cell-free reaction in batch mode<br/>The reaction mixture was prepared as described before [20].<br/>The reaction buffer is composed of: 50 mM Hepes pH 8, 1.5 mM<br/>ATP and GTP, 0.9 mM CTP and UTP, 0.2 mg/ml tRNA, 0.26 mM<br/>coenzyme A, 0.33 mM NAD, 0.75 mM cAMP, 0.068 mM folinic acid,<br/>1 mM spermidine, 30 mM 3-PGA, 1 mM DTT, 2% PEG8000. A<br/>typical cell-free reaction is composed of 33% (volume) of E. coli<br/>crude extract. The other 66% of the reaction volume are composed<br/>of the plasmids and the reaction buffer containing the nutrients.<br/>The amino acid concentration was adjusted to either 1.5 mM or<br/>3 mM of each of the 20 amino acids. Mg-glutamate and K-glutamate<br/>concentrations were adjusted according to the plasmids used<br/>(60 mM K-glutamate and 5 mM Mg-glutamate for pBEST-OR2-<br/>OR1-Pr-UTR1-deGFP-T500, 20 mM K-glutamate and 5 mM Mgglutamate<br/>for the T7 cascade). The concentration of synthesized<br/>deGFP was determined from a calibration curve (Ex 485 nm and<br/>Em 528 nm) done with pure recombinant eGFP (reGFP) purchased<br/>from Cell Biolabs Inc. End-point fluorescence was measured in a<br/>384 format well plate using a BioTek plate reader. deGFP synthesis<br/>kinetics were measured in a 96-well plate sealed with a plastic cap<br/>to avoid evaporation.





https://www.future-science.com/doi/full/10.2144/000114249

The 20 solid amino acids are dissolved at concentrations between 1.6 and 4 M in 5 M KOH, and mixtures containing each<br/>of the 20 amino acids are prepared for cell-free expression. The mixtures are diluted in water to achieve a total amino acid<br/>concentration between 253 and 268 mM. The pH of the resulting aqueous solutions is adjusted with acetic acid. The amino<br/>acid mixtures are frozen in liquid nitrogen and stored at -80°C.&nbsp;<span style="font-family: Roboto, sans-serif; font-size: 15px;">The 20 solid amino acids were dissolved in 5 M KOH to produce concentrations of 1.6–4 M. These stock solutions, stored at −20°C in a liquid phase, can be used for a few weeks.&nbsp;&nbsp;mixture stability depended on amino acid concentration, composition, and pH.&nbsp;On average, amino acid mixtures prepared with this method were 30 times less expensive than those prepared with an amino acid sampler kit.&nbsp;The mixtures were stable at room temperature (RT; ∼25°C), during aliquoting (∼20 min), and when snap frozen in liquid nitrogen for long storage at −80°C (∼6 months).&nbsp;</span><span style="font-family: Roboto, sans-serif; font-size: 14px;">deGFP was synthesized through the T7 cascade with 0.1 nM pBEST-OR2-OR1-Pr-UTR1-T7-T500 plasmid, 1 nM pIVEX2.3d-deGFP plasmid, 20 mM K-glutamate, and 2 mM Mg-glutamate.&nbsp;</span><span style="font-family: Roboto, sans-serif; font-size: 14px;">The synthesis was carried out using three different amino acid distributions: (A) constant, (B)&nbsp;</span>''E. coli''<span style="font-family: Roboto, sans-serif; font-size: 14px;">, and (C) deGFP sequence-based (1 mg/mL deGFP = 39.39 µM).&nbsp;&nbsp;</span>For the constant distribution (Figure 1A), the highest yield reached was 83 µM, which is equal to 2.11 mg/mL of active protein. This was obtained with a mixture containing ∼2.75 mM of each of the amino acids at pH 6.51. For the E. coli distribution, leucine is the most abundant amino acid (10.5%) and cysteine the least abundant (1.1%). The maximum deGFP synthesis was 1.55 mg/mL (∼61 µM) with amino acids stabilized at pH 6.04. The last concentration range was measured for the deGFP distribution mixture. Glycine (8.9%) and leucine (8.4%) were the most abundant, whereas cysteine (0.9%) and tryptophan (0.4%) were the least abundant. A protein yield of 1.35 mg/mL (∼53 µM) was measured at pH 6.82.&nbsp; L<span style="font-family: Roboto, sans-serif; font-size: 15px;">arger protein yields were reached at slightly acidic pHs between 6.0 and 6.8 and&nbsp;the highest production of active deGFP was observed with the constant distribution mixture</span><span style="font-family: Roboto, sans-serif; font-size: 14px;"></span><span style="font-family: Roboto, sans-serif; font-size: 15px;"></span>

The reporter protein deGFP, a slightly modified version of eGFP (10), was expressed through a transcriptional activation cascade using the following two plasmids (1): pBEST-p15A-OR2-OR1-Pr-T7rnap-T500 (0.1 nM) to express the T7 RNA polymerase and pIVEX2.3d-deGFP (1 nM)
<br/>
Briefly, BL21 Rosetta2 cells are washed with S30A buffer: 14 mM Mg-glutamate, 60 mM K-glutamate, 50 mM Tris, and 2 mM DTT, buffered with glacial acetic acid to pH 8.2. The cytoplasm is extracted using a cell-press (12000 LB) and clarified by centrifugation at 30,000 × g. Then the cellular extract is dialyzed at 4°C for 1 h against the S30B buffer (14 mM Mg-glutamate and 150 mM K-glutamate, buffered to pH 8.2 with 2 M Tris) using cassettes with a 10 kDa cut-off. The crude extract represents 33% (30 µL) of the final reaction volume (90 µL), giving roughly 9.2 mg/mL of protein, and the remaining volume has the following components: water, 2 mM Mg-glutamate, 20 mM K-glutamate, 2% PEG-8000 (v/v), amino acids, maltodextrin at 216.186 g/L, and the reaction buffer (RB). The RB in the cell-free reactions has the following components: 50 mM Hepes pH 8, 1.5 mM ATP and GTP, 0.9 mM CTP and UTP, 0.2 mg/mL tRNA, 30 mM 3-PGA, 0.26 mM CoA, 0.33 mM NAD, 0.75 mM cAMP, 0.068 mM folinic acid, and 1 mM spermidine

L-Amino acids

[https://www.sigmaaldrich.com/catalog/product/sial/laa21?lang=en&region=US https://www.sigmaaldrich.com/catalog/product/sial/laa21?lang=en&region=US], $<span style="color: rgb(77, 77, 77); font-family: Arial, Helvetica, sans-serif; font-size: 12px; text-align: right;">383</span>

4 amino acids: (trans-4-hydroxy-l-proline, L-cysteine hydrochloride, L-histidine hydrochloride, L-lysine hydrochloride) should be replaced by:&nbsp;L-proline Sigma 81709–25G,&nbsp; L-cysteine Sigma 30089–25G,&nbsp; L-histidine Sigma 53319–25G, L-lysine Sigma L5501–5G

&nbsp;&nbsp;Table S1 Amino acid preparation at equimolar concentrations.&nbsp;



{|
|-
| width="64" | 
| width="64" | MW
| width="64" | mM
| width="64" | µL AA mix
| width="64" | mM in KOH mixture
|-
| Alanine
| 89.09
| 4089
| 13.6
| 146
|-
| Arginine*
| 210.67
| 2314
| 22.2
| 135
|-
| Asparagine
| 132.12
| 3759
| 13.6
| 134
|-
| Asparticacid
| 133.1
| 3752
| 13.6
| 134
|-
| Cysteine
| 121.16
| 2465
| 22.2
| 143
|-
| Glutamicacid
| 147.13
| 3655
| 13.6
| 130
|-
| Glutamine
| 146.15
| 2501
| 22.2
| 145
|-
| Glycine
| 75.07
| 4210
| 13.6
| 150
|-
| Histidine
| 155.15
| 3281
| 13.6
| 117
|-
| Isoleucine
| 131.18
| 3765
| 13.6
| 134
|-
| Leucine
| 131.18
| 2549
| 22.2
| 148
|-
| Lysine
| 146.19
| 2392
| 22.2
| 139
|-
| Methionine
| 149.21
| 2491
| 22.2
| 145
|-
| Phenylalanine
| 165.19
| 1716
| 34
| 153
|-
| Proline
| 115.13
| 3883
| 13.6
| 138
|-
| Serine
| 105.9
| 3953
| 13.6
| 141
|-
| Threonine
| 119.12
| 3853
| 13.6
| 137
|-
| Tryptophan
| 204.23
| 1661
| 34
| 148
|-
| Tyrosine
| 181.19
| 2396
| 22.2
| 139
|}

All of the solids (LAA21–1KT) are purchased from Sigma-Aldrich (St. Louis, MO) and dissolved according&nbsp;to the concentration listed in the table above in 500 µL 5 M KOH solution

Note that the final concentration of the amino acid stocks solution is calculated to account for&nbsp;a volume increase due to the physical mass of the solid. As an estimation, the volume increases is half&nbsp;the mass of the solid.<br/>*Arginine monohydrocloride is dissolved in water. All of the other L-amino acids that have been used do&nbsp;not have counter ions.<br/>To 381.6 µL of equimolar amino acids mixture at 2807 mM in 5 M KOH, water is added up to ~4 mL,&nbsp;and the pH is adjusted with glacial acetic acid:<br/>• 100 µL acetic acid, pH = 8.49; the mixture is stable during aliquoting.<br/>• 105 µL acetic acid, pH = 7.86; the mixture is stable during aliquoting.<br/>• 110 µL acetic acid, pH = 6.51; the mixture is stable during aliquoting.

Table S2. Amino acids preparation according to the E. coli distribution (4)



{| cellspacing="0" cellpadding="0"
|-
| AA
| E. coli distribution&nbsp;%<br/>
| mM in KOH mixture<br/>
| µL AA mix<br/>
|-
| Alanine
| 9.7
| 280
| 26
|-
| height="19" | Arginine
| 5.3
| 154
| 25
|-
| height="19" | Asparagine
| 3.6
| 104
| 11
|-
| height="19" | Aspartic acid
| 4.8
| 139
| 14
|-
| height="19" | Cysteine
| 1.1
| 31
| 5
|-
| height="19" | Glutamic acid
| 5.4
| 156
| 16
|-
| height="19" | Glutamine
| 6.2
| 177
| 27
|-
| height="19" | Glycine
| 7.7
| 222
| 20
|-
| height="19" | Histidine
| 1.9
| 54
| 6
|-
| height="19" | Isoleucine
| 5.5
| 158
| 16
|-
| height="19" | Leucine
| 10.8
| 311
| 47
|-
| height="19" | Lysine
| 4.3
| 125
| 20
|-
| height="19" | Methionine
| 2.9
| 84
| 13
|-
| height="19" | Phenylalanine
| 4.1
| 119
| 27
|-
| height="19" | Proline
| 4.2
| 122
| 12
|-
| height="19" | Serine
| 5.5
| 158
| 15
|-
| height="19" | Threonine
| 5.1
| 146
| 14
|-
| height="19" | Tryptophan
| 1.5
| 44
| 10
|-
| height="19" | Tyrosine
| 2.7
| 78
| 12
|-
| height="19" | Valine
| 7.5
| 215
| 32
|}



All of the solids (LAA21–1KT) are purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved according to the concentrations listed in the table below in 500 µL 5 M KOH solution purchased from Sigma-Aldrich. Amino acids are dissolved in 5 M KOH as described in Supplementary Table S1.

*Arginine monohydrocloride is dissolved in water. All the others L-amino acids that have been used do not have counter ions.

To 369 µL of E. coli amino acids mixture at 2878 mM in 5 M K(OH), water is added up to ~4 mL, and the pH adjusted with glacial acetic acid:

• 95 µL acetic acid, pH = 8.21; the mixture is stable during aliquoting.

• 100 µL acetic acid, pH = 7.51; the mixture is stable during aliquoting.

• 110 µL acetic acid, pH = 6.04; the mixture is stable during aliquoting.



Table S3. Amino acids mixture preparation according to deGFP sequence.&nbsp;

{| border="0" cellspacing="0" cellpadding="0"
|-
| AA
| deGFP distribution&nbsp;%<br/>
| mM in K(OH) mixture&nbsp;<br/>
| &nbsp;µL AA mix<br/>
|-
| Alanine
| 3.7
| 105
| 10
|-
| height="19" | Arginine
| 2.6
| 73
| 12
|-
| height="19" | Asparagine
| 5.5
| 156
| 16
|-
| height="19" | Aspartic acid
| 7.2
| 203
| 21
|-
| height="19" | Cysteine
| 0.9
| 26
| 4
|-
| height="19" | Glutamic acid
| 5.7
| 162
| 17
|-
| height="19" | Glutamine
| 3.7
| 105
| 16
|-
| height="19" | Glycine
| 9.5
| 267
| 24
|-
| height="19" | Histidine
| 3.3
| 94
| 11
|-
| height="19" | Isoleucine
| 5.1
| 142
| 14
|-
| height="19" | Leucine
| 8.9
| 249
| 37
|-
| height="19" | Lysine
| 7.9
| 223
| 36
|-
| height="19" | Methionine
| 2.3
| 64
| 10
|-
| height="19" | Phenylalanine
| 5.8
| 162
| 36
|-
| height="19" | Proline
| 4.3
| 122
| 12
|-
| height="19" | Serine
| 4
| 113
| 11
|-
| height="19" | Threonine
| 6.5
| 184
| 18
|-
| height="19" | Tryptophan
| 0.4
| 12
| 3
|-
| height="19" | Tyrosine
| 4.4
| 123
| 20
|-
| height="19" | Valine
| 8.2
| 230
| 34
|}

The composition is retrieved from the gene sequence using [http://www.expasy.org/ http://www.expasy.org/]. All of the solids (LAA21–1KT) are purchased from Sigma-Aldrich (St. Louis, MO) and dissolved according to the concentrations listed in the table below in 500 µL 5 M KOH solution purchased from Sigma-Aldrich. Amino acids are dissolved in 5 M KOH as described in Supplementary Table S1.

*Arginine monohydrocloride is dissolved in water. All of the other L-amino acids that have been used do not have counter ions.

To 360 µL of the E. coli amino acids mixture at 2812 mM in 5 M K(OH), water is added up to ~4 mL, and the pH is adjusted with glacial acetic acid:

• 95 µL acetic acid, pH = 8.72; the mixture is stable during aliquoting.

• 100 µL acetic acid, pH = 7.9; the mixture is stable during aliquoting.

• 105 µL acetic acid, pH = 6.82; the mixture is stable during aliquoting.

== Protocol 7 ==

=== In Vitro Translation of scFv Antibody Fragments. ===

In vitro translation in an E. coli S-30 system was performed according to Chen and Zubay (16) with several modifications. Translation was carried out for 10 min at 37°C in a 110-μl reaction mixture that contained the following components: 50 mM Tris⋅HOAc at pH 7.5, 30 mM NH4OAc, 12.3 mM Mg(OAc)2, 0.35 mM each amino acid, 2 mM ATP, 0.5 mM GTP, 1 mM cAMP, 0.5 mg/ml E. coli tRNA, 20 μg/ml folinic acid, 100 mM KOAc, 30 mM acetylphosphate (17), 1.5% polyethylene glycol 8000, 33 μg/ml rifampicin, 1 mg/ml vanadyl ribonucleoside complexes (VRC), 23 μl of E. coli MRE600 extract (16), and 90 μg/ml mRNA.

=== Affinity Selection of Ribosome Complexes and Isolation of RNA. ===

The translation was stopped by adding Mg(OAc)2 to a final concentration of 50 mM and cooling on ice (18). The samples were diluted 4-fold with ice-cold washing buffer [50 mM Tris⋅HOAc, pH 7.5/150 mM NaCl/50 mM Mg(OAc)2/0.1% Tween 20] and centrifuged for 5 min at 4°C at 10,000 × g to remove insoluble components. Microtiter plates coated with hemagglutinin-transferrin conjugate were prewashed with ice-cold washing buffer, and the supernatant from the centrifuged translation mixture was applied (200 μl per microtiter well) and the plate was gently shaken for 1 hr in the cold room. After five washes with ice-cold washing buffer, the retained ribosome complexes were dissociated with ice-cold elution buffer (100 μl per well; 50 mM Tris⋅HOAc at pH 7.5/150 mM NaCl/10 mM EDTA/50 μg/ml E. coli tRNA) for 10 min in the cold room, and released mRNA was recovered by precipitation with ethanol or by isolation using the Rneasy kit (Qiagen).

=== Reverse Transcription–PCR and in Vitro Transcription. ===

Reverse transcription was performed using Superscript reverse transcriptase (GIBCO/BRL) according to the supplier’s recommendation. PCR was performed using Taq DNA polymerase (GIBCO/BRL) in the presence of 5% (vol/vol) dimethyl sulfoxide (4 min at 94°C, followed by 3 cycles of 30 sec at 94°C, 30 sec at 37°C, and 2 min at 72°C, followed by 10 similar cycles at 60°C instead of 37°C, 20 similar cycles at 60°C instead of 37°C with elongation at 72°C prolonged by 15 sec per cycle and finished by 10 min at 72°C). PCR products were analyzed by agarose gel electrophoresis and purified from the gel and reamplified, if the amount and quality was not sufficient, or directly used for transcription without additional purification. In vitro transcription was performed as described (19).

=== In Vitro Coupled Transcription–Translation and RIA. ===

After the fifth round of ribosome display, PCR products were cloned into the vector pTFT74 (20). In vitro coupled transcription–translation in the S-30 E. coli system was performed using 50 μg/ml plasmid DNA and conditions similar to those described above with the following modifications. A coupled transcription–translation was carried out for 30 min at 37°C, and the reaction mixture was supplemented with T7 RNA polymerase at 2000 units/ml and 0.5 mM UTP and TTP. The mixture contained [35S]methionine at 50 μCi/ml (1 μCi = 37 kBq) and each amino acid except methionine at 0.35 mM. After translation, the reaction mixture was diluted 4-fold with PBS and bound to immobilized hemagglutinin peptide in a microtiter well. After 60 min of incubation with gentle shaking, microtiter wells were washed five times with PBS containing 0.1% Tween 20, and bound radioactive protein was eluted with 0.1 M triethylamine. Eluted protein was quantified in a scintillation counter.
[[Category:Antibodies]]