Synthetic and Systems Biotechnology 4 (2019) 204–211

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Synthetic and Systems Biotechnology

journal homepage: http://www.keaipublishing.com/synbio

Methods to reduce variability in E. Coli-based cell-free protein expression T

experiments
Jared L. Dopp, Yeong Ran Jo, Nigel F. Reuel∗

A R T I C LE I N FO A B S T R A C T

Keywords: Cell-free protein synthesis (CFPS) is an established biotechnology tool that has shown great utility in many
Cell-free protein synthesis applications such as prototyping proteins, building genetic circuits, designing biosensors, and expressing cyto-
CFPS toxic proteins. Although CFPS has been widely deployed, the many, varied methods presented in the literature
Cell extract can be challenging for new users to adopt. From our experience and others who newly enter the field, one of the
In vitro protein synthesis
most frustrating aspects of applying CFPS as a laboratory can be the large levels of variability that are present
In vitro transcription-translation
Cell-free synthetic biology
within experimental replicates. Herein we provide a retrospective summary of CFPS methods that reduce
variability significantly. These methods include optimized extract preparation, fully solubilizing the master mix
components, and careful mixing of the reaction. These have reduced our coefficient of variation from 97.3% to
1.2%. Moreover, these methods allow complete novices (e.g. semester rotation undergraduate students) to
provide data that is comparable to experienced users, thus allowing broader participation in this exciting re-
search area.

1. Introduction experiments [25]. Commercial kits (such as Promega S30 T7 High-

Yield, PURExpress) that standardize reagent production can improve
Cell-free protein synthesis (CFPS) is an established biotechnology variation [26], but careful methods are also necessary to reduce
tool, used over 50 years ago to decipher the relationship between variability. In this methods paper, we summarize the evolution of best
mRNA codons and amino acid sequences [1]. It has had a recent re- practices that have improved the variability in our CFPS reactions such
naissance due to many new applications such as producing protein that the variability is comparable with a commercial kit (Promega S30
therapies [2–4], prototyping proteins [5], engineering metabolic net- T7 High-Yield - Fig. 1).
works [6,7], and biosensors [8–10]. Additionally, the composition of
cell extract from different strains has been analyzed including TB3, 2. Materials and methods
BL21 Star™ (DE3), A19, and BL21 Rosetta2 [11–14]. Many of the
components needed for CFPS (cell extract, supplements and additives) All chemical reagents were purchased from Sigma Aldrich unless
are now commercially available. This has led many groups, including otherwise noted.
our own, to adopt CFPS as a convenient tool for in-house expression of
custom proteins. This adoption is evident in a rapid increase in re- 2.1. Cell growth and extract preparation
viewed papers that use E. coli based CFPS [15]. There are many good
reviews on CFPS to orient new users on basic methods, current uses, Our process to develop scalable methods to improve cell extract
and potential future applications [5,16–20]. Despite this rapid adoption with reduced variability is published in detail [26] and outlined here
and widespread information, CFPS can still be difficult to implement, (Fig. 2). The E. coli strain BL21 Star™ (DE3) (Invitrogen) is used due to
especially for new users, due to variability that stems from the small its rapid doubling time, T7 transcription machinery, and ability to ex-
reaction volumes and sensitive reagents. Multiple recent papers have press proteins from linear DNA due to a mutation in the RNaseE gene
pointed out this issue of variability [21–24]. There has also been some (rne131) [28,29]. This strain of E. coli is induced with IPTG during
work aimed at identifying and quantifying interlaboratory variability growth to produce T7 RNA polymerase (T7RNAP) for the cell-free re-
[22]. In fact, the National Institute of Standards and Technology (NIST) action. This is useful when the genetic template utilizes a T7 promoter
recently held a workshop to address the sources of variability in CFPS as it eliminates the need to supplement T7RNAP to the extract post

Peer review under responsibility of KeAi Communications Co., Ltd.

∗
Corresponding author.
E-mail address: [email protected] (N.F. Reuel).

https://doi.org/10.1016/j.synbio.2019.10.003
Received 2 July 2019; Received in revised form 21 October 2019; Accepted 24 October 2019
2405-805X/ © 2019 Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
J.L. Dopp, et al. Synthetic and Systems Biotechnology 4 (2019) 204–211

2.1.2. Day 2 (cell growth and harvest)

6. Add 100 mL 1 M glucose to the 900 mL 2x YTP to make 1L of 2x

YTPG and warm in the shaker incubator for 30 min at 37 °C.
7. If an optimum growth time has already been determined, inoculate
the 2x YTPG with the 20 mL starter culture (step 5 above).
Otherwise, generate a new growth curve using OD600 measure-
ments taken at a regular time interval (15–30 min). First, remove
20 mL of the 2x YTPG to use as a baseline reference in the spec-
trophotometer. Then, inoculate the growth media with the 20 mL
starter culture.
8. Significant: Once the desired point of induction has been reached
(acquired via an earlier designed experiment campaign [26]) add
1 mL of 1 M IPTG. For the BL21 Star™ we have previously de-
termined this to be 3 h 20 min for 1 L growths in 2.5L Tunair flasks
Fig. 1. Reporter protein, sfGFP, expression kinetics obtained by our group
[26] shaken at 270 rpm.
showing progression of methods to reduce variability. ‘Initial Methods’ were
our first attempts at expression using a PANOx-SP system [27], ‘intermediate
methods’ refer to our implementation of Sutro's modified Cytomim system [4],
Note: Optimizing cell growth in this way is meant to improve yield
‘current methods’ refer to improved techniques (detailed herein) using the while also reducing the number of measurements taken by the re-
PANOx-SP system, and the Promega S30 T7 High-Yield Protein Expression kit is searcher. In contrast, recent literature shows that optimization may not
a commercial kit used for benchmarking against these methods. The shaded be necessary when using cell-free autoinduction (CFAI) media. This
area represents 1 standard deviation (n = 4). offers another viable option for those who wish to reduce variability
caused by manual steps as much as possible [32].
growth, which can be another source of variance. Optimal conditions
for IPTG exposure and total growth time were determined and reported 9. Harvest at the desired overall growth time. We have determined
in terms of the extent of the cell growth curve (as optical density me- this to be 4 h 15 min for 1 L growths in 2.5 L Tunair flasks [26].
trics are not absolute and can change based on instrument and vessel Distribute the growth evenly among centrifuge tubes and weigh
used). If a new cell line is used, a designed experiment (DoE) should be each. Ensure the mass of all tubes is identical by adding ddH2O.
used to determine optimal growth conditions (induction and harvest 10. Centrifuge the cell suspension at 5000 xg and 4 °C for 15 min to
times) as these can have a large impact on protein expression efficiency pellet the cells.
of the extract [26,30]. However, it may be of interest to note that ob- 11. Decant the supernatant and collect the cell pellets in a 50 mL
served variability in CFPS performance is not significantly impacted by conical tube using a metal spatula. Keep on ice
extract preparation as long as protocols are followed faithfully [22]. 12. Resuspend the cell pellet in cold buffer A via vortexing.
The combined cell growth and extract preparation process is by far the 13. Centrifuge the cell pellet at 5000 xg and 4 °C for 10 min to pellet the
most laborious and time intensive; it is presented here as daily steps. cells.
14. Discard the supernatant and wipe any residual liquid from inside
2.1.1. Day 1 (media and buffer preparation) the tube using a Kimwipe.
15. Significant: Record the mass of the cells. This is referred to as the
1. Prepare and autoclave 500 mL of LB media (5 g tryptone, 2.5 g yeast wet cell mass. Under these conditions, a wet cell mass of 5–7 g is
extract, 5 g sodium chloride, and ddH2O to 500 mL) typically obtained from each liter.
2. Prepare 900 mL of 2x YTP media (16 g tryptone, 10 g yeast extract,
5 g sodium chloride, 7 g potassium phosphate dibasic, 3 g potassium Optional: Freeze the cell pellet and store at −80 °C for later use.
phosphate monobasic, and ddH2O to 900 mL). Titrate the 2x YTP to The pellet can be flash frozen in liquid N2 or frozen in the freezer with
pH 7.2 using 5 M KOH and autoclave. no noticeable detrimental effects downstream. This step is optional
3. Significant: Prepare a 500 mL solution of 1 M glucose and filter because the cells can be lysed immediately after harvesting, if desired.
using a Nalgene™ Rapid-Flow™ sterile disposable filter unit with PES
membrane (ThermoFisher #166-0045). Glucose will eventually be 2.1.3. Day 3 (cell lysis and extract lyophilization)
added to the 2x YTP media in order to produce 2x YTPG. We choose There are two methods we commonly use to disrupt the cell mem-
to filter the glucose to avoid a Maillard reaction which has been brane: sonication and French press homogenization. When lysing small
shown to impair media performance [31]. This can also be avoided amounts of cells (< 25 g wet cell mass) it is more efficient to use a
by autoclaving 2xYTP and glucose in separate containers. sonication protocol. However, large amounts (≥25 g wet cell mass) are
4. Prepare and filter a 500 mL solution of buffer A (10 mM tris-acetate best handled with homogenization [26].
pH 8.2, 14 mM magnesium acetate, and 60 mM potassium acetate).
5. Once the LB media has cooled to room temperature, aliquot 20 mL 2.1.3.1. Option 1: French press homogenization (preferred)
into a 50 mL conical tube and add a cell pick of BL21 Star™ (DE3).
Allow this starter culture to grow overnight (14–18 h) at 37 °C and 17. Add 1 mL buffer A and 1 mL ddH2O per gram wet cell mass to the
230 rpm in a MaxQ 4000 shaker (Thermo Scientific). We have found pellet. Resuspend the pellet via vortexing. The buffer and the extra
that the quantity of the cell pick and this timing do not have to be water lower the viscosity of this mixture enough to eliminate
exact as the overnight cultures double to the point of resource ex- homogenizer clogging and maintain a more consistent pressure
haustion and thus enter the cell growth in Day 2 with a similar swing of the homogenizer piston. The excess water will be driven
concentration of cells (little difference in growth curves observed). off later through lyophilization.
Allow the 2x YTP to set at room temperature overnight. 18. Turn on the vacuum pump and the homogenizer (Avestin
EmulsiFlex C3). Clean the homogenizer by flushing it 3 times with
water and a 70% (v/v) ethanol solution. Repeat 3 times with
ddH2O to ensure removal of all ethanol.
19. Prime the homogenizer with ddH2O and put on standby so it

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Fig. 2. High level overview of the cell growth and extract preparation process. Steps in the process are grouped into categories based on which day the steps are
performed.

doesn't run dry. Only keep a small amount of water in the hopper so the protein concentration achieved using other lysing methods (i.e.
as not to dilute the cells any more than necessary. sonication) or other desired concentrations.
20. Significant: Place the metal heat exchanger coil located in the
outlet tube in a beaker of ice to facilitate cooling. 27. Freeze the diluted extract at −80 °C for 30 min.
20. Pour the cell suspension into the hopper and start the machine. 28. Significant: Prepare the 35 L VirTis Genesis Pilot-scale Lyophilizer
21. Ensure there is a clean beaker to collect the resulting crude lysate. (SP Scientific) by setting the shelf temperature to 15 °C. A smaller,
22. Maintain a pressure swing of 25,000–30,000 psi by continuously bench top lyophilization unit that sublimates in vessels at room
adjusting the pressure knob (again this can only be done if the cells temperature can also be used, but we have found better extract
are diluted, if the mixture is too thick the swing is inconsistent and performance from extract that has been lyophilized at a chilled
the homogenizer will stall). shelf temperature.
29. Place the baking pan in the lyophilizer and turn on the vacuum. The
Note: The above conditions have been optimized for use with the vacuum should be set to 200 mTorr.
homogenizer in our lab. By implementing the same method (i.e. using a 28. Turn on the condenser and allow it to lyophilize overnight.
designed experiment), the optimum for any membrane disruption Optional: Depending on the volume being lyophilized, the neces-
method can be determined. sary water loss can occur in as little as 4 h [26].

23. Upon completion, collect the crude lysate in 5 mL microcentrifuge

2.1.4. Day 4 (extract reconstitution and storage)
tubes.
24. Centrifuge the crude lysate at 12,000 xg and 4 °C for 10 min.
29. Follow the lyophilization shutdown procedure by turning off the
25. Decant the supernatant into a 50 mL conical tube and place on ice.
condenser, then turn off the vacuum, then turn on the vacuum
release.
Optional: It is historically common to perform a run-off reaction at
30. Once the internal pressure has equilibrated, transfer the contents of
this point to improve protein yields by digesting any remaining nucleic
the baking pan to a pre-weighed 50 mL conical tube and record the
acids with endogenous enzymes. This is typically done in small volumes
mass. The resulting powder is fluffy and has a charge, so it is best to
(~500 μL) around 37 °C and 250 rpm for an optimized amount of time
crush the powder with a spatula before transferring to the tube.
[16,33]. However, certain E. coli strains such as BL21 Star™ (DE3)
produce a robust extract that does not require a runoff reaction [23,34].
Optional: Store the lyophilized extract for later use. Lyophilized
To optimize protein yield, it may be necessary to optimize new strains
extract can remain active at elevated temperatures (−20 °C or 4 °C) for
or promoter systems [34,35]. Because of this, we do not outline them in
at least a year [36].
this protocol. The extract will then need to be centrifuged and collected
as in steps 24 and 25.
31. Add water to the lyophilized extract according to the following
Optional: The extract can be dialyzed to remove metabolic by-
equation ddH2O (mL) = (mass of lyophilized extract (g))(1.5 mL/
products. This is typically performed with using 10K MWCO cutoff
0.094 g) and allow to sit at room temperature for 5 min before re-
cassette multiple times with chilled buffer A. This step has been popular
suspension. Resuspend by pipetting up and down.
in the past but has become less common [23,26,34]. Dialysis may also
32. Significant: Use PCR tubes to store the extract in 45 μL aliquots at
be necessary to optimize new strains and promoter systems [34,35].
−80 °C. While this takes a large amount of time, it saves reagent
The extract will then need to be centrifuged and collected as in steps 24
and avoids multiple freeze thaw cycles. The aliquots will depend on
and 25.
personal preferences but 45 μL works well since each 15 μL reaction
has 3.6 μL. This is enough extract to reliably support 2 sets of 5
26. Prepare the dilute extract for lyophilization by placing it in a
replicates.
container that will provide a large surface area and good heat
transfer (a metal baking pan works well). Cover the pan with a
large Kimwipe and tape it to the pan. 2.1.5. Day 3 (alternative method)
2.1.5.1. Option 2: sonication. Sonication is suitable for small amounts of
Note: Lyophilization, traditionally, is an optional step that may or cell lysis (< 25g wet cell mass). When developing a sonication protocol,
may not meet the needs of the user. In this case, the extract we produce designed experiments can test parameters such as amplitude, energy,
using a French press is dilute, so we perform lyophilization to remove and sample volume. This allows for the possibility of a more controlled
the excess water. In this way, we can reconstitute our extract to match lysing environment for extract since settings on piston-based
continuous homogenizers cannot be as rigorously controlled.

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However, the throughput of the continuous homogenizer will always be templates to prototype the proteins. We have previously published the
greater when large volumes of extract need to be processed [23,26,34]. development process, advantages, and next steps for this method [5].
Optional: Turn on the cooling system for the sonicator. The cooling Here we outline the methods:
system helps prevent sample to sample variation by keeping a more
consistent sample temperature than a typical ice bath. 1. A minimal genetic template is designed to contain primer binding
sites, restriction enzyme sites, a T7 promoter, a ribosome binding
17. Add 1 mL buffer A per gram wet cell mass to the pellet. Resuspend site, a start codon, and a T7 terminator. The desired amino acid is
the pellet via vortexing. codon optimized using an online tool (IDT Codon Optimization
18. Transfer the suspension to 1.5 mL microcentrifuge tubes in 1 mL Tool) [38,39] and is encoded between the start and end codon.
aliquots and keep on ice. 2. The gene fragment can then be ordered from a vendor of choice (e.g.
19. Set the sonicator (Qsonica Q125 with a 2 mm (5/64 in) tip) to 50% IDT, Twist).
amplitude and 10 s on/off pulse. 3. Once the gene fragment has been received, it is suspended and un-
20. Significant: Sonicate the samples, one by one, on ice (if not using a dergoes traditional linear amplification. We use both GoTaq
cooling system) until an energy input of 532 J has been reached. (Promega) and OneTaq (New England BioLabs) with no observed
This number was determined by previously optimized methods differences. If there are concerns about template length (> 1.5 kb) a
[23]. This takes about 5 min per sample. Place each sample on ice product like Phusion (New England BioLabs) is more precise in
post sonication. amplification. Protocols supplied by the vendor are followed in this
21. Centrifuge the crude lysate at 12,000 xg and 4 °C for 10 min. step. Promega and New England BioLabs (NEB) both have online
22. Using a 200 μL pipette carefully transfer the supernatant to a 50 mL melting temperature (Tm) calculators that are dependent on the
conical tube. Gently homogenize the clarified extract to ensure any product and the primers used for amplification.
batch to batch variability is eliminated. 4. The resulting linear template (LET) is then purified (using Zymo Kit
#D4003) and eluted in at least 50 μL of buffer (Qiagen EB #19086).
Optional: Run-off reactions, dialysis, and lyophilization steps after If desired, the LET can be quantified, but 45 μL must be reserved for
this are the same as detailed in the homogenizer section above. the restriction digest.
5. Restriction digest is performed by adding 5 μL of Cut Smart buffer
23. Significant: Use PCR tubes to store the extract in 45 μL aliquots at (NEB) and 1 μL (20 units) restriction enzyme (HindIII-HF from NEB
−80 °C. While this takes a large amount of time, it saves reagent #R3104S) to the 45 μL of LET. Run the digestion for 1 h at 37 °C and
and avoids multiple freeze thaw cycles. then inactivate the enzyme at 80 °C for 20 min.
6. After digestion, the “sticky ends” from the digest are ligated together
2.3. DNA amplification to form a small circle template using T4 (or T7) ligase (New England
Biolabs). This is done by adding 5 μL T4 DNA Ligase buffer (NEB
One of the advantages of CFPS is that it can use both plasmids and #M0202S) and 2 μL T4 (800 units) DNA ligase to the digested
polymerase chain reaction (PCR) products directly as expression tem- product and run at room temperature for 1 h.
plates [5,17,37]. We have found both to work equally well and can be 7. The minimal circular expression template (CET) is then purified
made in house. Plasmids are useful once desired sequences are settled (using Zymo Kit #D4003) and eluted in 45 μL of buffer (Qiagen EB
on as they can be transformed into cells and then harvested to create #19086).
large stocks of DNA template. PCR products are much more useful in 8. Rolling circle amplification can work on DNA concentrations in the
the prototyping phase, as the work stream is much shorter (no trans- pg/μL range, but it is not necessary to dilute the DNA to this low of a
formation of cells) but they are more expensive to produce and the DNA concentration. Typically, we add 1–2 μL of DNA from the purified
yield is orders of magnitude smaller than by cells. We have recently CET stock to 100 μL of double distilled water as our dilute stock for
demonstrated how rolling circle amplification (RCA) can be used to isothermal RCA.
mitigate this problem by amplifying a small amount of mail-order gene
fragment to large amounts of DNA for protein expression [5]. NOTE: The dilute CET is only meant for a one-time use. Do not store
it for extended periods (> 1 week at −20 °C) as we find its performance
2.3.1. Plasmids suffers in later CFPS experiments.
The pJL1-sfGFP plasmid was used for all data in this manuscript
[23]. Plasmids are amplified using the following steps. 9. Rolling circle amplification (RCA) is performed using a TempliPhi™
kit (GE Healthcare). The kit comes with 3 solutions: a lysis buffer
1. After cloning the gene of interest into the plasmid and transforma- (red cap), amplification buffer (blue cap), and phi29 (Φ29) poly-
tion into E. coli, colonies containing the plasmid are grown and merase (yellow cap). It is best to aliquot master mixes of this for
stored in a glycerol stock. future amplifications to reduce the number of freeze cycles. For one
2. This glycerol stock is then used for subsequent growth harvest for group of RCA reactions, mix 20 μL lysis buffer, 20 μL reaction
DNA purification. buffer, and 0.8 μL Φ29 polymerase. Add 4 μL of dilute CET (from
3. The cells are grown, lysed, and the DNA is purified using ZymoPURE step 8 above) and distribute evenly among 4 PCR tubes (11.2 μL per
II MaxiPrep Kit (Zymo Research); this kit is selected for purification tube). According to the protocol from the manufacturer, the reac-
due to the column design. The column has a narrow neck which tion is exhausted after 4 h at 30 °C. To parallelize lab work, we tend
allows for smaller volume elution and higher concentration pur- to let this reaction run overnight and purify the following morning.
ifications, if desired. The instructions provided with the kit are 10. The resulting product is diluted with 14 μL of double distilled
followed exactly except a different elution buffer that does not water, purified (using Zymo Kit #D4003), eluted in at least 25 μL of
contain EDTA (such as Qiagen EB #19086) must be used since EDTA buffer (Qiagen EB #19086), quantified, and stored at −20 °C for
is known to chelate magnesium. later use in CFPS. A high-level overview of the process is shown in
4. DNA is then quantified using a NanoPhotometer® N80 (Implen). Fig. 3.

2.3.2. Rolling circle amplification 2.4. Master mix preparation

As our lab relies on new, custom proteins for downstream applica-
tions, we use the aforementioned RCA method to quickly create genetic There are many recipes of supplements to add to the CFPS reaction.

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Fig. 3. Simplified overview of the rolling circle

amplification from minimal template process. The
entire process takes less than 24 h and results in a
large amount of genetic temple that is suitable for
CFPS.

We have previously used the modified Cytomim system outlined by and store in 60 μL aliquots at −80 °C. Stock solutions are thawed on
Sutro Biopharma but found the low yield to be troublesome for small- ice but can also be thawed more quickly on a thermo block at 30 °C
scale research applications [4,26]. One that we have found to be most if necessary. Upon thawing, the solutions should be vortexed for
productive and least variable is the PANOx-SP recipe [28]. Our varia- homogeneity.
tion of the PANOx-SP “master mix” includes: HEPES, ATP, GMP, CMP,
UMP, phosphoenolpyruvate (PEP), folinic acid, E. coli tRNA, 20 amino The variability in our experiments was drastically reduced when we
acids (purchased from Formedium, United Kingdom), NAD, CoA, po- started using completely soluble master mix. To illustrate, the PANOx-
tassium glutamate, ammonium glutamate (MP Biomedicals), magne- SP (final) sample in Fig. 1 is a fully soluble master mix that is on par
sium glutamate, potassium oxalate, putrescine, and spermidine. Since with the master mix provided by Promega's S30 T7 High-Yield CFPS kit
we induce the production of T7RNAP during cell growth, there is no (#L1110). The initial sample in Fig. 1 were not completely solubilized
need to add to the master mix. The best practices for the development of and the increased variance is apparent. Despite this, there is an ad-
a master mix are as follows: vantage to splitting the master mix into multiple mixtures in order to
calibrate magnesium ion concentration. Each batch of cell extract is
1. Significant: Create concentrated stock mixes of each of these different and requires a different magnesium concentration in the final
components which can then be combined to form the master mix reaction to optimize protein production [16,27,33,35]. This step of
solution. Creating a stock solution for most of these components is creating a custom master mix, we have found, is not critical but should
relatively straightforward since most of them are soluble in water. It be done with large batches of extract that will be used for long periods
is important to note that some are only partially soluble in water; of time. It should also be noted that observed variability is heavily in-
these take some additional steps. fluenced by the preparation of the master mix [22]. As such, we believe
2. Significant: To prepare a 1 M PEP solution, one must use a com- this is the most critical step in CFPS.
bination of water and KOH until PEP is completely dissolved. Titrate
the solution to a pH of 7 with KOH.
2.5. Cell-free reaction
3. Significant: The most difficult stock to make is the amino acid so-
lution. Our stock solution is 50 mM of 20 amino acids and not all are
Finally, in setting up the cell-free reactions there are also key steps
readily soluble at neutral pH. Tyrosine, in particular, will not dis-
to reduce variability. A CFPS reaction requires only 4 components: cell
solve even when heated. While it has been reported that a non-
extract, DNA template, master mix, and water to bring the solution to
homogenous amino acid suspension is not detrimental to CFPS
final reaction volume. Final reaction volumes are typically small
[4,27], we prefer to completely solubilize our amino acid mixture
(< 20 μL) and there are two ways to create sample replicates (Fig. 4).
using previously reported methods [40]. This reduces the chance of
Method 1 involves aliquoting each small volume into separate wells, or
having insoluble fragments of the amino acids present in reactions.
tubes, and homogenizing each reaction individually. These experi-
The amino acids are dissolved in a mixture of water and 5 M KOH
mental replicates include the error introduced by pipetting small
until a pH of 12 is achieved. The pH of the solution can be adjusted
sample volumes. Method 2 involves adding all the components into a
by adding acetic acid. It is important to note that amino acids will
single mixture tube, homogenizing, and then aliquoting the replicates
start to drop out of solution if the pH drops below 12. The amino
from this mixture into separate wells. This method eliminates the error
acids should also be added in order of most hydrophobic to least
introduced by small volume pipetting errors as each are derived from
hydrophobic, and completely solubilized before adding the next
the same mixture and the combined volumes are larger and easier to
amino acid. While a stock amino acid solution pH of 12 may seem
manage by standard pipettes. In this manner the end variability mea-
high, the buffering capacity of the master mix is maintained when
sured is caused solely by differences in CFPS expression variation and
all components are combined. We find the final pH of the master
not pipetting. One drawback to this method is that it requires slightly
mix is 7.0–7.3.
more reagent than desired. For example, in order to conduct 5 re-
4. Spermidine may come as a liquid. If this is the case, do not make a
plicates, we typically mix the necessary volume for 6 replicates due to
stock, just use the specific gravity to determine how much should be
loss in pipetting and wetting of plastic surfaces. We have also noticed
added to the final master mix. This is done in the spreadsheet pro-
that the reduction of pipetting error from Method 2 allows CFPS novices
vided in the supplement.
to perform experiments with reduced variance (Fig. 5). The master mix
5. Determine the final concentration for each component in the master
used to generate Fig. 5 replaces ATP with AMP; which is used to reduce
mix. This is typically done by setting up a spreadsheet and working
cost in training, but also results in > 3x reduction in expression effi-
backwards from needed final concentrations (see example sheet in
ciency (comparing Figs. 1–5).
Supplement 1). Set the cell extract and genetic template con-
The composition of our typical cell-free reaction are as follows:
centrations, then determine what volume of the final reaction is
57 mM HEPES (pH 7), 1.2 mM ATP, 0.85 mM GMP, 0.85 mM CMP,
reserved for the master mix. For instance, our CFPS reactions are
0.85 mM UMP, 33 mM PEP, 34 μg/mL folinic acid, 171 μg/mL E. coli
typically 15 μL total volume with 5 μL reserved for the master mix.
tRNA, 2 mM 20 amino acids, 0.33 mM NAD, 0.26 mM CoA, 175 mM
In the provided spreadsheet, the variables typically manipulated
potassium glutamate, 10 mM ammonium glutamate, 16 mM magnesium
(DNA concentration, number of replicates, etc.) are highlighted in
glutamate, 2.7 mM potassium oxalate, 1 mM putrescine, 1.5 mM sper-
green.
midine, 13.3 ng/μL pJL1-sfGFP plasmid DNA, and 24% (v/v) cell ex-
6. A supplement mix can be stored as separate solutions (salts, energy,
tract. The components were added to a 1.5 mL tube (DNA added last)
PEP, etc.) [27,33] or all components can be stored together into one
and homogenized before being aliquoted to wells in 15 μL aliquots. Due
master mix. We combine all components into a single tube, vortex,
to the variability of small CFPS reactions, ours are always carried out in

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Fig. 4. Two methods of preparing sample replicates. In Method 1, components for each reaction are directly aliquoted to the individual reactors, then subsequently
homogenized. In Method 2, all components are homogenized first in a mixture tank and then aliquoted as complete reaction replicates to single containers.

n = 4 or 5 samples in order to obtain better population sampling. When times. The total volume should be 90 μL so the pipette should be set
using the Promega kit, the ratio of master mix to total reaction volume to 45 μL.
in the manufacturer's protocol is maintained when scaling down the 5. Pipette the reaction mix into 5 individual wells in 15 μL aliquots.
reaction. To keep results comparable, we use previously optimized 6. Cover the plate with film and run under desired reaction conditions.
extract [26]. The reactions are typically carried out at 37 °C in a 384
black-walled, flat-bottom well plate (Greiner #781906) and covered We use a plate reader to measure sfGFP since we believe activity is
with a colorless film (Axygen). Fluorescence measurements are taken the most objective measurement of correctly folded protein. Other
with a Synergy Neo2 HTS Multi-Mode Microplate Reader (BioTek) for proteins (enzymes, antibodies, etc.) may require more specific assays
at least 4 h. In cases where expression longer than 4 h is desired, the (binding, kinetics, toxicity, etc.) to determine their quality. In these
temperature is reduced to 30 °C. The fluorescence of sfGFP is measured cases, it may be best to assess titer through purification. In this way,
at an excitation of 485 nm and emission of 528 nm using a ± 20 activity per concentration can be used to objectively assess the quality
bandpass window and 61 gain setting. The reaction is also stirred in of the process.
orbital motion at 237 cpm; although it is disputed whether mixing is
needed for such small volumes, we find it does improve sample
homogeneity [28]. To summarize the reaction procedure: 3. Results and discussion

1. Determine the amount of extract, DNA, master mix, and ddH2O The data presented in Figs. 1 and 5 show how CFPS results can have
needed for six 15 μL reactions. reduced variability with improved methods. The initial trials (Fig. 1)
2. Thaw, on ice, the amount of reagent needed to run all samples and used the PANOx-SP system [23] but without concern for completely
controls. dissolving all the master mix components and using replicate Method 1
3. In a 1.5 mL microcentrifuge tube, add in the following order: in Fig. 4. The final trials (Fig. 1) also used PANOx-SP master mix but
ddH2O, extract, master mix, and DNA. While we see no observed following the methods described in this work. Since the average signal
significance in the order of addition, DNA should always be added of each sample between these experiments is different, it's best to
last since it starts the reaction. compare the variability between these samples using the coefficient of
4. Homogenize the reaction mix by pipetting up and down at least 10 variation (CV) or relative standard deviation, which is computed by
dividing the standard deviation by the mean value and expressed as a

Fig. 5. Comparison of data obtained from the two

replicate generating methods mentioned in Fig. 4,
where A) is obtained from mixing all reaction
components individually in wells (includes pipet-
ting error) and B) involves mixing all reactants in a
single mixture tube first, then aliquoting the mix-
ture replicates to independent wells (n = 5). Shaded
error region is for one standard deviation. Note: the
master mix used in these training experiments uses
AMP instead of ATP to further reduce cost [41], but
comes at a loss of yield (compare to Fig. 1 which
used a modified ATP based master mix).

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J.L. Dopp, et al. Synthetic and Systems Biotechnology 4 (2019) 204–211

Fig. 6. Significant method steps in the preparation

of a cell-free reaction that can reduce variability and
increase efficiency. A) keeping a filtered stock of
1 M glucose for cell growth, B) storing cell extract in
45 μL aliquots for single use, C) homogenizing all
reactants in a tube before aliquoting into wells, and
D) creating a fully solubilized master mix.

percentage. We find the CV of the initial attempt to be 97.3% and was production scale the master mix composition and cell lysis method
improved to 1.2% using these methods. This is comparable to the would be driven by cost factors. Thus, the methods outlined herein are
commercial kit (Promega #L1110) CV of 1.14%. When comparing the targeted at the researcher using CFPS for fast protein prototyping where
data obtained from an experienced user to a novice (undergrad re- small yields (μg-mg) are sufficient.
searcher with limited experience in CFPS), method 1 of replicate pre-
paration results in CVs of 6.8% and 11.0%. When method 2 of replicate 4. Conclusions
preparation is used, there is a noticeable decrease in variance from both
experimentalists, with CVs of 3.5% and 3.0% for experienced and no- In conclusion, the variability that can be observed in CFPS experi-
vice respectively. This also achieves some level of standardization so ments, especially in runs performed by new users, can be reduced sig-
results between team members can be compared. nificantly by adopting the methods presented in this paper. Of the
We believe the small difference in performance and variation be- method steps we highlight in this paper, the most significant are 1)
tween the Promega master mix and our own (Fig. 1) is likely due to completely dissolving all master-mix components for their individual
makeup of the commercial master mix. The use of nucleoside tripho- stock solutions, 2) making enough master mix to last the entire ex-
sphates (GTP, CTP, UTP) may provide a performance advantage over perimental campaign, and 3) homogenizing all reactants before ali-
the less expensive monophosphates (GMP, CMP, UMP) used in our quoting replicates to reduce the small-volume pipetting error (Fig. 6).
master mix [27]. It could also be due to differences in reagent con- In this manner the researcher can obtain more statistically significant
centration. The Promega documentation does not state the concentra- results and those new to the field of in vitro expression can more reliably
tions of their master mix components, therefore a true comparison is contribute to CFPS experimental campaigns. This is of importance as
difficult to make. Despite the lack of information, we do observe that CFPS moves from a niche tool to a technique used by larger teams for
the variability using both these mixes is comparable when following the biomaterial, metabolic network, and sensor discovery and design.
remainder of the methods presented in this work.
While it is impossible to completely eliminate all sources of varia- Acknowledgements
tion, following the procedures outlined here, we have found, reduces
experimental error. Machine errors such as unequal heating, shaking We would like to acknowledge Bradley Bundy, Michael Jewett,
and measurement error will remain. Other sources can be attributed to James Swartz, Vincent Noireaux, and Yongchan Kwon for their assis-
the stochastic nature of biological processes. It is also important to note tance in understanding and discussing these protocols. NFR acknowl-
that the methods outlined in this paper are for the expression of sfGFP edges funding from the Black & Veatch Building a World of Difference
but the methods used may need to be further optimized for other pro- Faculty Fellowship in Engineering and Iowa State University Startup
teins of interest. For example, expressing various immunoglobulins Funds.
required different temperatures for optimum expression [42]. There-
fore, the process should be optimized for the protein(s) of interest, if Appendix A. Supplementary data
maximum expression is desired.
If CFPS is being used for larger batch production (such as in dis- Supplementary data to this article can be found online at https://
tributed manufacturing scenarios [43]) or large scale production (such doi.org/10.1016/j.synbio.2019.10.003.
as for cytotoxic therapies [2]), many of these sources of variability (e.g.
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