Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Summary

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Abstract

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 µg/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per µg of protein produced or $0.019 per µL of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Introduction

Cell-free Protein Synthesis (CFPS) has emerged as a technology that has unlocked a number of new opportunities for protein production, functional genomics, metabolic engineering, and more within the last 50 years^1,2. Compared to standard in vivo protein expression platforms, CFPS provides three key advantages: 1) the cell-free nature of the platform enables the production of proteins that would be potentially toxic or foreign to the cell^3,4,5,6; 2) inactivation of genomic DNA and the introduction of a template DNA encoding the gene(s) of interest channel all of the systemic energy within the reaction to the production of the protein(s) of interest; and 3) the open nature of the platform enables the user to modify and monitor the reaction conditions and composition in real time^7,8. This direct access to the reaction supports the augmentation of biological systems with expanded chemistries and redox conditions for the production of novel proteins and the tuning of metabolic processes^2,9,10. Direct access also allows the user to combine the CFPS reaction with activity assays in a single-pot system for more rapid design-build-test cycles¹¹. The capacity to perform the CFPS reaction in small volume droplets or on paper-based devices further supports high-throughput discovery efforts and rapid prototyping^{12,13,14,15,16}. As a result of these advantages and the plug and play nature of the system, CFPS has uniquely enabled a variety of biotechnology applications such as the production of proteins that are difficult to solubly express in vivo^17,18,19,20, detection of disease^21,22,23, on demand biomanufacturing^{18,24,25,26,27}, and education^28,29, all of which show the flexibility and utility of the cell-free platform.

CFPS systems can be generated from a variety of crude lysates from both prokaryotic and eukaryotic cell lines. This allows for diverse options in the system of choice, each of which have advantages and disadvantages depending on the application of interest. CFPS systems also vary greatly in preparation time, cost, and productivity. The most commonly utilized cell extracts are produced from wheat germ, rabbit reticulocyte, insect cells, and Escherichia coli cells, with the latter being the most cost-effective to date while producing the highest volumetric yields of protein³⁰. While other CFPS systems can be advantageous for their innate post-translational modification machinery, emerging applications using the E. coli-based machinery are able to bridge the gap by generating site-specifically phosphorylated and glycosylated proteins on demand^{31,32,33,34,35}.

CFPS reactions can be run in either batch, continuous-exchange cell-free (CECF) or continuous-flow cell-free (CFCF) formats. The batch format is a closed system whose reaction lifetime is limited due to diminishing quantities of reactants and the accumulation of inhibitory byproducts of the reaction. CECF and CFCF methods increase the lifetime of the reaction, and thereby result in increased volumetric protein yields compared to the batch reaction. This is accomplished by allowing the byproducts of protein synthesis to be removed from the reaction vessel while new reactants are supplied throughout the course of the reaction². In the case of CFCF, the protein of interest can also be removed from the reaction chamber, while in CECF, the protein of interest remains in the reaction chamber comprised of a semi-permeable membrane^36,37. These methods are especially valuable in overcoming poor volumetric yields of difficult-to-express proteins of interest^{38,39,40,41,42,43}. The challenges in implementing the CECF and CFCF approaches are that 1) while they result in more efficient use of the bio machinery responsible for transcription and translation, they require notably larger quantities of reagents that increases overall cost and 2) they require more complex reaction setups and specialized equipment compared to the batch format⁴⁴. In order to ensure accessibility for new users, the protocols described herein focus on the batch format at reaction volumes of 15 µL with specific recommendations for increasing the reaction volume to the milliliter scale.

The methods presented herein enable non-experts with basic laboratory skills (such as undergraduate students) to implement cell growth, extract preparation, and batch format reaction setup for an E. coli-based CFPS system. This approach is cost-effective compared to commercially available kits without sacrificing the ease of kit-based reaction setup. Furthermore, this approach enables applications in the laboratory and in the field. When deciding to implement CFPS, new users should thoroughly evaluate the efficacy of conventional protein expression systems for startup investment, as CFPS may not be superior in every case. The CFPS methods described here enable the user to directly implement a variety of applications, including functional genomics, high-throughput testing, the production of proteins that are intractable for in vivo expression, as well as field applications including biosensors and educational kits for synthetic biology. Additional applications such as metabolic engineering, tuning of protein expression conditions, disease detection, and scale-up using CECF or CFCF methods are still possible but may require experience with the CFPS platform for further modification of reaction conditions. Our methods combine growth in enriched media and baffled flasks, with relatively rapid and reproducible methods of cell lysis through sonication, followed by a simplified CFPS reaction setup that utilizes optimized premixes⁴⁵. While the cellular growth methods have become somewhat standardized within this field, methods for cell lysis vary widely. In addition to sonication, common lysis methods include utilization of a French press, a homogenizer, bead beaters, or lysozyme and other biochemical and physical disruption methods^46,47,48,49. Using our methods, approximately 2 mL of crude cell extract are obtained per 1 L of cells. This quantity of cell extract can support four hundred 15 µL CFPS reactions, each producing ~900 µg/mL of reporter sfGFP protein from the template plasmid pJL1-sfGFP. This method costs $0.021/µg of sfGFP produced ($.019/µL of reaction), excluding the cost of labor and equipment (Supplemental Figure 1). Starting from the scratch, this method can be implemented in 4 days by a single person and repeat CFPS reactions can be completed within hours (Figure 1). Additionally, the protocol can be scaled up in volume for larger batches of reagent preparation to suit the user's needs.Importantly, the protocol presented here can be implemented by laboratory trained non-experts such as undergraduate students, as it only requires basic laboratory skills. The procedures described below, and the accompanying video have been specifically developed to improve accessibility of the E. coli CFPS platform for broad usage.

Protocol

1. Media Preparation and Cell Growth

1. Day 1
   1. Streak E. coli BL21*(DE3) cells from a glycerol stock onto an LB agar plate and incubate for at least 18 h at 37 °C.
   2. Prepare 50 mL of LB media and autoclave the solution on a liquid cycle for 30 min at 121 °C. Store at room temperature.
2. Day 2
   1. Prepare 750 mL of 2x YTP media and 250 mL of 0.4 M D-Glucose solution as described in the supplemental information.
   2. Pour the 2x YTP media into an autoclaved 2.5 L baffled flask and the D-Glucose solution into an autoclaved 500 mL glass bottle. Autoclave both solutions on a liquid cycle for 30 min at 121 °C.
   3. Ensure that both sterile solutions are stored at 37 °C if cell growth is being performed on the next day, to maximize growth rates upon inoculation. Do not combine solutions until inoculation.
      NOTE: Solutions can be stored at 4 °C for 1-2 d if needed, though the 2x YTP media is highly prone to contamination.
   4. Start an overnight culture of BL21(DE3) by inoculating 50 mL of LB media with a single colony of BL21(DE3) using a sterilized loop and sterile technique to avoid contamination.
   5. Place the 50 mL of BL21*(DE3) LB culture into a 37 °C 250 rpm shaking incubator and grow overnight for 15-18 h.
   6. Prepare and sterilize all materials required for days 3 and 4, including: two 1 L centrifuge bottles, 4x cold 50 mL conical tubes (weigh and record masses of three), and many 1.5 mL microfuge tubes.
3. Day 3
   1. Remove the 50 mL overnight culture of BL21*(DE3) in LB from the shaking incubator and measure the OD₆₀₀ on a spectrophotometer using a 1:10 dilution with LB media. Calculate the volume of overnight culture necessary to add to 1 L of media for a starting OD₆₀₀ of 0.1 (For example, if an OD₆₀₀ of a 1:10 dilution is read as 0.4, inoculate 25 mL of the undiluted OD₆₀₀ = 4.0 overnight culture into 1 L of 2x YTPG).
   2. Remove the warmed 2x YTP media and D-Glucose solutions from the 37 °C incubator along with the 50 mL of LB culture. Using sterile technique, carefully pour the D-Glucose solution into the 2x YTP media (avoiding the sides of the baffled flask).
      NOTE: Addition of D-Glucose completes the recipe for 1 L of 2x YTPG.
   3. Maintaining sterile technique, inoculate the 1 L of 2x YTPG solution with the appropriate amount of the 50 mL culture to begin the 1 L culture at a 0.1 OD₆₀₀. Immediately place the inoculated 1 L culture into a 37 °C shaking incubator at 200 rpm.
   4. Take the first OD₆₀₀ reading after the first hour of growth (lag phase typical takes 1 h). Do not dilute the culture. Continue taking OD₆₀₀ measurements approximately every 20-30 min until OD₆₀₀ reaches 0.6.
   5. Upon reaching OD₆₀₀ = 0.6, add 1 mL of 1M IPTG (final concentration in 1 L culture = 1 mM) to the 2x YTPG culture.
      NOTE: Ideal induction OD₆₀₀ is 0.6; however, a range of 0.6-0.8 is acceptable. Induction by IPTG is for endogenous production of T7 RNA Polymerase (T7RNAP).
   6. After induction, measure the OD₆₀₀ approximately every 20-30 min until it reaches 3.0.
      NOTE: Cool down the centrifuge to 4 °C during this time. Prepare cold S30 buffer as detailed in the Supplementary Information. If the S30 buffer is prepared in advance, ensure that DTT is not added until the day of use.
   7. Once the OD₆₀₀ reaches 3.0 (Figure 2A), pour the culture into a cold 1 L centrifuge bottle in an ice-water bath. Prepare a water-filled 1 L centrifuge bottle of equal weight to be used as a balance in the centrifuge.
      NOTE: Absorbance values vary from instrument-to-instrument. While the OD₆₀₀ of harvest of BL21(DE3) is not a sensitive variable, it is recommended that the user evaluate and optimize this variable as a troubleshooting measure. Larger spectrophotometers may result in relatively lower OD₆₀₀ readings compared to smaller cuvette-based spectrophotometers.
   8. Centrifuge the 1 L bottles for 10 min at 5,000 x g and 10 °C to pellet cells.
   9. Slowly pour off the supernatant and dispose of it according to the institution's biological waste procedures. Place the pellet on ice.
   10. Using a sterile spatula, scrape the cell pellet from the centrifuge bottle and transfer it to a cold 50 mL conical tube.
   11. Add 30 mL of cold S30 buffer to the conical tube and resuspend the cell pellet by vortexing with short bursts (20 - 30 s) and rest periods (1 min) on ice until fully resuspended with no chunks.
   12. Once the pellet is fully resuspended, use another 50 mL conical tube with water as a balance and centrifuge for 10 min at 5000 x g and 10 °C (pre-cooled to 4 °C).
       NOTE: This completes the 1^st of 3 washes required when harvesting the cells.
   13. Pour out the supernatant and dispose of it according to the institution's biological waste procedures. Resuspend the pellet with 20-25 mL of cold S30 buffer and centrifuge for 10 min at 5000 x g and 10 °C (pre-cooled to 4 °C).
       NOTE: This completes the 2^nd of 3 washes.
   14. Again, pour out the supernatant and dispose of it according to the institution's biological waste procedures. Add exactly 30 mL of S30 buffer and vortex again to resuspend the pellet.
   15. Using the 3 pre-weighed, cold 50 mL conical tubes and a serological pipette filler with a sterile pipette, aliquot 10 mL of resuspended pellet/S30 buffer mixture into each of the 3 conical tubes.
       NOTE: Splitting the cells into 3 tubes is not required, but this step results in smaller cell pellets (~ 1 g) for increased convenience at later steps.
   16. Centrifuge all tubes, using appropriate balances as needed, for 10 min at 5000 x g and 10 °C (pre-cooled to 4 °C).
       NOTE: This completes the final wash step.
   17. Pour out the supernatant and dispose of it according to the institution's biological waste procedures. Remove the excess S30 buffer by carefully wiping the inside of the conical tube and cap with a clean tissue; avoid touching the pellet.
   18. Reweigh the tubes on an analytical balance and record the final pellet weight on each tube.
       NOTE: The protocol can be paused at this point. The pellets can be flash frozen in liquid nitrogen and stored at -80 °C for up to a year until needed for extract preparation.

2. Crude Cell Extract Preparation - Day 4

1. For extract preparation, keep cells cold on ice during each step. Add 1 mL of cold S30 buffer per 1 g of cell mass of the pellet. Ensure that dithiothreitol (DTT) has been supplemented to the S30 buffer to a final concentration of 2 mM.
   NOTE: Cool down the microcentrifuge to 4 °C during this time.
2. Resuspend the cell pellet by vortexing with short bursts (20 - 30 s) and rest periods (1 min) on ice until fully resuspended. If resuspension is difficult, leave the pellets on ice for 30 min to defrost.
3. Transfer 1.4 mL of resuspended cells into a 1.5 mL microfuge tube.
4. Place one 1.5 mL tube containing 1.4 mL of resuspended cells into an ice water bath in a beaker. Sonicate for 45 s on followed by 59 s off for 3 total cycles, with amplitude set at 50%. Close and invert the tubes to gently mix during the off periods. In total, deliver 800-900 J of energy to each 1.5 mL microfuge tube containing 1.4 mL of resuspended cells (Figure 3A & 3B).
   NOTE: This step is sensitive to the sonicator type and model used and should be optimized if equipment is different than listed for this procedure. Two complementary approaches can be used to scale-up the amount of extract prepared during this step: 1) multiple 1.5 mL microfuge tubes can be sonicated in parallel, and/or 2) larger volumes can be sonicated in conical tubes (up to 15 mL of cell resuspension per tube), scaling the amount of energy delivered as previously described ^29,45.
5. Immediately after sonication is complete, add 4.5 µL of 1 M DTT (supplementing an additional 2 mM DTT) into the 1.4 mL of lysate and invert several times to mix. Place the tube on ice. Repeat steps 2.4 and 2.5 for any additional tubes of resuspended cells before proceeding to centrifugation.
6. Microcentrifuge samples at 18,000 x g and 4 °C for 10 min (Figure 3C).
7. Pipette the supernatant into a new 1.5 mL microfuge tube. Do not disturb the pellet; it is preferable to leave some supernatant behind to maintain purity than to disrupt the pellet in efforts to maximize yield.
8. Incubate the supernatant from the previous step at 250 rpm and 37 °C for 60 min by taping the tubes to the shaking platform of the incubator (this is the runoff reaction).
9. Microcentrifuge samples at 10,000 x g and 4 °C for 10 min.
10. Remove the supernatant without disturbing the pellet and transfer it to a new tube. Create many 100 µL aliquots of extract for storage.
    NOTE: The protocol can be paused here, and the extract can be flash frozen in liquid nitrogen and stored at -80 °C for up to a year until needed for CFPS reactions. At least 5 freeze-thaw cycles can be undergone without detriment to extract productivity (Figure 4).

3. Cell-Free Protein Synthesis Batch Format Reactions

1. Thaw Solutions A and B, DNA template, BL21(DE3) extract (if frozen), T7RNAP, and an aliquot of molecular grade water.
   NOTE: CFPS reaction template can be found in the Supplementary Information. Solutions A and B recipes are provided in the Supplementary Information and correspond to specific concentrations for numerous reagents to support the PANOx-SP based energy system for CFPS. The role of each reagent and acceptable variation in these reagent concentrations that can support CFPS have been determined⁵⁰. A T7RNAP purification protocol can be found in the Supplementary Information⁵¹. Supplemental T7RNAP can increase volumetric yields but is not necessary if T7RNAP is induced during cell growth. Plasmid DNA template (pJL1-sfGFP) can be prepared using a maxiprep kit with two washes using the wash buffer in the kit, followed by a post-processing DNA-cleanup using a PCR purification kit (Figure 2B). Linear DNA templates can also be used in CFPS reactions.
2. Label the necessary amount of microfuge tubes needed for CFPS reactions.
   NOTE: Reactions can be performed in various vessel sizes, but a smaller vessel can decrease volumetric protein yields (Figure 2C). Scaling up a reaction in the same size vessel may also reduce volumetric yields, as a function of decreasing the oxygen exchange, due to a decrease in the surface area to volume ratio. When increasing reaction volume above 100 μL, it is recommended to use flat bottom well plates ^31,37,52.
3. Add 2.2 µL of Solution A, 2.1 µL of Solution B, 5 µL of BL21*(DE3) extract, 0.24 μg of T7RNAP (16 μg/mL final concentration), 0.24 ng of DNA template (16 ng/µL final concentration), and water to bring the final volume to 15 µL.
   NOTE: Vortex Solutions A and B frequently during reaction setup to avoid sedimentation of components and ensure that each reaction receives a homogenous aliquot of each solution. Avoid vortexing the extract, instead invert the tube to mix.
4. After all reagents have been added to the reaction, mix each tube by pipetting up and down or gently vortexing while ensuring that the final reaction mixture is combined into a single 15 µL bead at the bottom of the 1.5 mL microfuge tube.
5. Place each reaction into the 37 °C incubator without shaking for 4 h, or 30 °C overnight.
   NOTE: Successful reactions can be qualitatively assessed visually based on the green color of the sfGFP product within the CFPS reaction mixture (Figure 3D). Expression of the protein of interest can also be confirmed by SDS-PAGE (Supplemental Figure 2).

4. Quantification of the Reporter Protein, [sfGFP]

1. Load 48 µL of 0.05 M HEPES, pH 8, into each well needed for quantification (usually performed in triplicate per reaction tube).
2. Remove reactions from incubator. Pipette up and down to mix each reaction, then transfer 2 µL of reaction into the 48 µL of 0.05 M HEPES, pH 8. Pipet up and down again in the well to mix.
3. Once all reactions are loaded and mixed, place the 96 well plate into the fluorometer and measure the sfGFP endpoint fluorescence.
   NOTE: Excitation and emission wavelengths for sfGFP fluorescence quantification are 485 nm and 510 nm, respectively.
4. Using a previously generated standard curve, determine the [sfGFP] from the obtained fluorescence readings.
   NOTE: Instructions for generating a standard curve of sfGFP concentration versus fluorescence intensity are provided in Supplementary Information (Supplementary Figure 3). Users will need to establish a standard curve for their instrument since instrument sensitivity may vary.

Results

We have presented a sonication-based E. coli extract preparation protocol that can be completed over a four-day span, with Figure 1 demonstrating the procedural breakdown over each day. There is malleability to the steps that can be completed in each day with various pausing points, but we have found this workflow to be the most effective to execute. Additionally, both the cell pellets (step 1.3.18) and fully prepared extract (step 2.10) are stable a...

Discussion

Cell-free protein synthesis has emerged as a powerful and enabling technology for a variety of applications ranging from biomanufacturing to rapid prototyping of biochemical systems. The breadth of applications is supported by the capacity to monitor, manipulate, and augment cellular machinery in real-time. In spite of the expanding impact of this platform technology, broad adaptation has remained slow due to technical nuances in the implementation of the methods. Through this effort, we aim to provide simplicity and cla...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Luria Broth	ThermoFisher	12795027
Tryptone	Fisher Bioreagents	73049-73-7
Yeast Extract	Fisher Bioreagents	1/2/8013
NaCl	Sigma-Aldrich	S3014-1KG
Potassium Phosphate Dibasic	Sigma-Aldrich	60353-250G
Potassium Phosphate Monobasic	Sigma-Aldrich	P9791-500G
D-Glucose	Sigma-Aldrich	G8270-1KG
KOH	Sigma-Aldrich	P5958-500G
IPTG	Sigma-Aldrich	I6758-1G
Mg(OAc)2	Sigma-Aldrich	M5661-250G
K(OAc)	Sigma-Aldrich	P1190-1KG
Tris(OAc)	Sigma-Aldrich	T6066-500G
DTT	ThermoFisher	15508013
tRNA	Sigma-Aldrich	10109541001
Folinic Acid	Sigma-Aldrich	F7878-100MG
NTPs	ThermoFisher	R0481
Oxalic Acid	Sigma-Aldrich	P0963-100G
NAD	Sigma-Aldrich	N8535-15VL
CoA	Sigma-Aldrich	C3144-25MG
PEP	Sigma-Aldrich	860077-250MG
K(Glu)	Sigma-Aldrich	G1501-500G
NH4(Glu)	MP Biomedicals	02180595.1
Mg(Glu)2	Sigma-Aldrich	49605-250G
Spermidine	Sigma-Aldrich	S0266-5G
Putrescine	Sigma-Aldrich	D13208-25G
HEPES	ThermoFisher	11344041
Molecular Grade Water	Sigma-Aldrich	7732-18-5
L-Aspartic Acid	Sigma-Aldrich	A7219-100G
L-Valine	Sigma-Aldrich	V0500-25G
L-Tryptophan	Sigma-Aldrich	T0254-25G
L-Phenylalanine	Sigma-Aldrich	P2126-100G
L-Isoleucine	Sigma-Aldrich	I2752-25G
L-Leucine	Sigma-Aldrich	L8000-25G
L-Cysteine	Sigma-Aldrich	C7352-25G
L-Methionine	Sigma-Aldrich	M9625-25G
L-Alanine	Sigma-Aldrich	A7627-100G
L-Arginine	Sigma-Aldrich	A8094-25G
L-Asparagine	Sigma-Aldrich	A0884-25G
Glycine	Sigma-Aldrich	G7126-100G
L-Glutamine	Sigma-Aldrich	G3126-250G
L-Histadine	Sigma-Aldrich	H8000-25G
L-Lysine	Sigma-Aldrich	L5501-25G
L-Proline	Sigma-Aldrich	P0380-100G
L-Serine	Sigma-Aldrich	S4500-100G
L-Threonine	Sigma-Aldrich	T8625-25G
L-Tyrosine	Sigma-Aldrich	T3754-100G
Fisherbrand Premium Microcentrifuge Tubes: 2.0 mL	Fisher Scientific	05-408-138
Fisherbrand Premium Microcentrifuge Tubes: 1.5 mL	Fisher Scientific	05-408-129
Fisherbrand Premium Microcentrifuge Tubes: 0.6 mL	Fisher Scientific	05-408-120
PureLink HiPure Plasmid Prep Kit	ThermoFisher	K210007
Ultrasonic Processor	QSonica	Q125-230V/50Hz	3.175 mm diameter probe
Avanti J-E Centrifuge	Beckman Coulter	369001
JLA-8.1000 Rotor	Beckman Coulter	366754
1L Centrifuge Tube	Beckman Coulter	A99028
Tunair 2.5L Baffeled Shake Flask	Sigma-Aldrich	Z710822
Microfuge 20	Beckman Coulter	B30134
New Brunswick Innova 42/42R Incubator	Eppendorf	M1335-0000
Cytation 5	BioTek
Strep-Tactin XT Starter Kit	IBA	2-4998-000
pJL1-sfGFP	Addgene	69496
BL21(DE3)	New England BioLabs