S30 E. coli extract

Protocol 1, S30 E. coli extract

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

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

2.2. Biomass Processing
1. S30 buffer, approximately 100 mL: 10 mM Tris acetate, 14 mM magnesium acetate, 60 mM potassium acetate ( see Note 6 ).
2. Avestin Emulsifl ex homogenizer.
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
the 20 amino acids, 105 mM phosphoenol pyruvate (PEP), 8.4 U/mL pyruvate kinase (Sigma-Aldrich #P7768).

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

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.
Alternatively, inoculate from a colony on an agar plate or a tube culture. Incubate the culture overnight at 37°C with
vigorous shaking (~250–280 rpm).
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-
Gauze fi lter lining in the cap ( see Note 2 ). Incubate at 37°C vigorous shaking (~250–280 rpm) and monitor growth by
OD at 600 nm.
3. Harvest the culture during exponential phase before the growth rate drops during the transition to stationary phase
( see Note 11 ). The culture should be chilled as quickly as possible either by adding ice directly to the culture or by passing it
through a heat exchanger or cooling coil in ice water.
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
be centrifuged at once, pour the supernatant off the cell pellets and add more culture on top the pellet for a second centrifugation.
Approximately 2.5 g/L/OD wet cells should be collected (~8 g from 1 L at 3 OD).
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.
6. Centrifuge the cell suspension at 8,000 × g for 20–30 min.
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
Note 13 ).
2. Shake and stir the cell suspension periodically until it is well mixed. Once thawed, keep the cell suspension on ice if the lysis
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
high pressure homogenizer at 17,500 psi ( see Note 14 ). The
lysate should be cooled as quickly as possible after exiting the
homogenizer, preferably through a cooling coil or heat
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
Note 15 ).

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

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

7. Centrifuge the extract at 4,000 × g for 10 min.

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

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

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

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

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

2. Mix reagents in the order listed in Table 1 , adding the plasmid
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
3MM filter paper for each reaction and label them with
pencil. Pierce each strip with a straight pin and stick the pin
in a sheet of Styrofoam covered in aluminum foil (shipping
cooler lids work well). Make sure the fi lter paper is held above
the foil by the pin so that the sample will not spread from
the fi lter to the foil.

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

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

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

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

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

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

4. Notes

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

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

3. The specifi c medium used for the inoculum is not critical, but
preferably it would be the same as the main culture medium.
However, in my experience, 2YT works better for overnight
inoculum cultures than 2YTPG. If 2YTPG is used for the inoculum,
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
used, but 2YTPG provides a good balance of cost, simplicity,
and reasonable cell density (which affects the yield of extract).
Also, 2YTPG will produce better extract than 2YT for longer
cell-free reactions ( 2 ) . The 2YTPG medium should be sterile
filtered instead of autoclaved to avoid carmelization of the glucose.
Alternatively, a separate 50% (v/v) glucose stock can be
made and added to the other medium components after autoclaving.
The growth rate of the culture determines the ribosomal
content of the extract ( 11 ) , so a medium which supports
rapid growth ( m > 0.5 h −1 ) should be used for best results. If
antifoam 204 is used, samples should be chilled to £ 25°C before
measuring the OD to eliminate the cloudiness of the antifoam.
Placing a 1 mL sample on ice for a couple minutes works well
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
vary depending on the strain and medium used. Metabolic
waste products generated from the glucose in the 2YTPG
medium will cause the culture to become acidic as the cell
density increases, so pH control can help sustain rapid growth
to higher cell densities. If pH control is not possible, high
quality extract can still be produced, but the culture may need
to be harvested at a lower cell density. In addition to pH control,
fed-batch fermentation methods can be used to reach
higher cell densities (thus producing more extract per batch),
but such methods are more complicated to set up and operate.
There are many published strategies for fed-batch fermentations
and a detailed description is beyond the scope of this
chapter. If a fed-batch method is employed, it is important that
it maintains a rapid growth rate to produce the highest ribosome
content in the extract ( 11 ) .

6. S30 buffer is typically prepared as needed from purifi ed water
and three separate 100× concentrates (1 M Tris acetate pH 8.2,
1.4 M magnesium acetate, and 6 M potassium acetate) which
are sterile fi ltered for increased stability and stored at room
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
with varying magnesium concentrations for testing the magnesium
optimum of each batch of extract. Typically, 8 mM magnesium
in the cell-free reaction (equivalent to 80 mM in the
10× salt solution) gives acceptable performance, but the optimum
may vary from about 4 to 12 mM. All reagents for extract
testing should be divided into small aliquots, frozen in liquid
nitrogen, and stored at −80°C for the greatest stability.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Recipe for a 15 μL cell-free transcription-translation reaction
Reagent Stock concentration Reaction concentration Volume ( μL)
Milli-Q water 5.5525
10× salt solution 10× 1× 1.5
10× master mix 10× 1× 1.5
AA mix 50 mM each 2 mM 0.6
Pyruvate 1 M 33 mM 0.5
Oxalate 1 M 4 mM 0.06
Putrescine 200 mM 1 mM 0.075
Spermidine 200 mM 1.5 mM 0.1125
CoA 20 mM 0.27 mM 0.2
NAD 100 mM 0.33 mM 0.05
14 C Leu 0.1 mCi/mL 1.7 μCi/mL 0.25
T7 RNA polymerase 0.8 mg/mL 33 μg/mL 0.6
Plasmid 0.5 mg/mL 13.3 μg/mL 0.4
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
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
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
Preparation of S30-T7 lysate

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

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

1780: pT7A3-T7-RNAPol-Y639F, 0.5 mM IPTG

1080 pBad24-T7-RNAPol: 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,  300.16 g Mg(OAc)2 Tetrahydrate (Sigma 228648)

6.0 M K(Acetate),  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

 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. 

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</span>600≈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.

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.

Cell-free reaction component	Final concentration in solution
HEPES-KOH (pH = 8)	55 mM
Magnesium acetate	14 mM
Potassium acetate	50 mM
Ammonium acetate	155 mM
Polyethylene glycol (PEG)	3% (v/v)
3-phosphoglycerate (3-PGA)	40 mM
20 amino acids	2.5 mM
Adenine triphosphate (ATP)	1.2 mM
Guanidine triphosphate (GTP)	1 mM
Uridine triphosphate (UTP)	0.8 mM
S30-T7 lysate	30% (v/v)
DNA template	10 pg/mL
DNase, RNase free H2O to reaction volume

A. Materials and solutions required: 

1M HEPES-KOH (pH = 8)

1M magnesium acetate

1M potassium acetate

5.2 M ammonium acetate

50% (v/v) Polyethylene glycol 6000 (PEG)

0.5 M 3-phosphoglycerate (3-PGA)

Notes: 

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.

100 mM Adenine triphosphate (ATP)

50 mM Guanidine triphosphate (GTP)

100 mM Uridine triphosphate (UTP)

S30-T7 lysate Prepared according to Table 1

DNase, RNase free H2O B. Equipment:

Notes: Floor incubator shaker or a Thermomixer® C. Procedure: 

Protocol 4

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

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

3.1.1. Day One: Buffer preparation and overnight culture 

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.

 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 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	Stock conc.	MW	Dry weight (g)	Stock volume (mL)	Final conc. in reaction mixture	Supplier	Storage (oC)
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		0.8 mM	Sigma
		523.18	0.131		0.8 mM	Sigma
		484.14	0.121		0.8 mM	Sigma
Total				10	0.8 mM		–80
Cyclic AMP	100 mM	351.2	0.351	10	0.64 mM	Sigma	–80
Folinic Acid	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		0.0175	1	0.175 mg/mL	Roche	–20
Creatine kinase	10 mg/mL	18 kDa	0.1	10	250 µg/mL	Roche	–80
T7 RNA polymerase					93 µg/mL		–20
pKO1166					32 µg/mL		–20
Plasmid					16 µg/mL		–20
S30 extract					~225 µL/mL		–80
Potasisum glutamate	4 M	203.23	40.65	50	208 mM	Sigma	–80
Magnesium acetate	1.07 M	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. 

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
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
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. 

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

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

Table 4: Components of a 500 µL reaction mixture and 5 mL outside buffer
Stock solution Reaction mixture (µL) Outside
buffer	(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. 

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) 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
using the BL21 Rosetta2 strain and with the following slight modifications.
S30A buffer composition: 14 mM Mg-glutamate, 60 mM
K-glutamate, 50 mM Tris buffered with acetic acid to pH 8.2. S30B
buffer composition: 14 mM Mg-glutamate, 150 mM K-glutamate
buffered to pH 8.2 with Tris. The lysate was prepared with a cell
press at a pressure of 16,000 LB.
2.2. Cell-free reaction in batch mode
The reaction mixture was prepared as described before [20].
The reaction buffer is composed of: 50 mM Hepes pH 8, 1.5 mM
ATP and GTP, 0.9 mM CTP and UTP, 0.2 mg/ml tRNA, 0.26 mM
coenzyme A, 0.33 mM NAD, 0.75 mM cAMP, 0.068 mM folinic acid,
1 mM spermidine, 30 mM 3-PGA, 1 mM DTT, 2% PEG8000. A
typical cell-free reaction is composed of 33% (volume) of E. coli
crude extract. The other 66% of the reaction volume are composed
of the plasmids and the reaction buffer containing the nutrients.
The amino acid concentration was adjusted to either 1.5 mM or
3 mM of each of the 20 amino acids. Mg-glutamate and K-glutamate
concentrations were adjusted according to the plasmids used
(60 mM K-glutamate and 5 mM Mg-glutamate for pBEST-OR2-
OR1-Pr-UTR1-deGFP-T500, 20 mM K-glutamate and 5 mM Mgglutamate
for the T7 cascade). The concentration of synthesized
deGFP was determined from a calibration curve (Ex 485 nm and
Em 528 nm) done with pure recombinant eGFP (reGFP) purchased
from Cell Biolabs Inc. End-point fluorescence was measured in a
384 format well plate using a BioTek plate reader. deGFP synthesis
kinetics were measured in a 96-well plate sealed with a plastic cap
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
of the 20 amino acids are prepared for cell-free expression. The mixtures are diluted in water to achieve a total amino acid
concentration between 253 and 268 mM. The pH of the resulting aqueous solutions is adjusted with acetic acid. The amino
acid mixtures are frozen in liquid nitrogen and stored at -80°C. 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.  mixture stability depended on amino acid concentration, composition, and pH. On average, amino acid mixtures prepared with this method were 30 times less expensive than those prepared with an amino acid sampler kit. 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). 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. The synthesis was carried out using three different amino acid distributions: (A) constant, (B) E. coli, and (C) deGFP sequence-based (1 mg/mL deGFP = 39.39 µM).  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.  Larger protein yields were reached at slightly acidic pHs between 6.0 and 6.8 and the highest production of active deGFP was observed with the constant distribution mixture

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)
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, $383

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

  Table S1 Amino acid preparation at equimolar concentrations. 

	MW	mM	µL AA mix	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 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 a volume increase due to the physical mass of the solid. As an estimation, the volume increases is half the mass of the solid.
*Arginine monohydrocloride is dissolved in water. All of the other L-amino acids that have been used do not have counter ions.
To 381.6 µL of equimolar amino acids mixture at 2807 mM in 5 M KOH, water is added up to ~4 mL, and the pH is adjusted with glacial acetic acid:
• 100 µL acetic acid, pH = 8.49; the mixture is stable during aliquoting.
• 105 µL acetic acid, pH = 7.86; the mixture is stable during aliquoting.
• 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)

AA	E. coli distribution %	mM in KOH mixture	µL AA mix
Alanine	9.7	280	26
Arginine	5.3	154	25
Asparagine	3.6	104	11
Aspartic acid	4.8	139	14
Cysteine	1.1	31	5
Glutamic acid	5.4	156	16
Glutamine	6.2	177	27
Glycine	7.7	222	20
Histidine	1.9	54	6
Isoleucine	5.5	158	16
Leucine	10.8	311	47
Lysine	4.3	125	20
Methionine	2.9	84	13
Phenylalanine	4.1	119	27
Proline	4.2	122	12
Serine	5.5	158	15
Threonine	5.1	146	14
Tryptophan	1.5	44	10
Tyrosine	2.7	78	12
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. 

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

The composition is retrieved from the gene sequence using 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.