From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Summary

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.

Abstract

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

Introduction

The use of cell-free protein synthesis (CFPS) for biotechnology applications has grown substantially over the past few years^1,2,3. This development can be attributed in part to increased efforts in understanding the processes that occur in CFPS and the role of each component^4,5. Additionally, reduced costs attributed to optimized set-ups and alternative energy sources have made cell-free technology easier to implement for new users^6,7,8,9. In order to implement the necessary transcription and translation factors for protein synthesis, cell extract is often used to drive cell-free reactions¹⁰. Recently published user guides have provided simple protocols for producing functional extract, making it easier to implement for new and experienced users alike^{1,11,12,13,14}. Cell extract is usually obtained through the lysis of a cell culture, which can be grown using different organisms depending on the specific use desired^1,15,16.

Escherichia coli (E. coli) has rapidly become one of the most commonly used host organisms for producing functional extracts¹⁷. The BL21 Star (DE3) strain is preferred because it removes the proteases from the outer membrane (OmpT protease) and the cytoplasm (Lon protease), providing an optimal environment for the recombinant protein expression. Additionally, the DE3 contains the λDE3 that carries the gene for T7 RNA polymerase (T7 RNAP) under the control of the lacUV5 promoter; the star component contains a mutated RNaseE gene which prevents cleavage of mRNA^4,14,18,19. Under the lacUV5 promoter, isopropyl-thiogalactopyranoside (IPTG) induction allows the expression of T7 RNAP^20,21. These strains are used to grow and harvest cells, which give raw material for extract preparation. Cell lysis can be performed using a variety of methods, including bead beating, French press, homogenization, sonication, and nitrogen cavitation^1,11,12,22.

The process of bacterial culture and harvesting is consistent across most platforms when using E. coli, but requires multiple days and intense researcher oversight^1,11,13. This process generally starts with an overnight seed culture in LB broth, which upon overnight growth is then inoculated into a larger culture of 2xYTPG (yeast, tryptone, phosphate buffer, glucose) the next day. The growth of this larger culture is monitored until it reaches the early-to-mid log phase, at an optical density (OD) of 2.5^14,20. Constant measurement is required as the components of transcription and translation have been previously demonstrated to be highly active in the early-to-mid log phase^23,24. While this process can create reproducible extract, our lab has recently developed a new method using Cell-Free Autoinduction (CFAI) Media, which reduces researcher oversight, increases the overall yield of extract for a given liter of cell culture, and improves access to E. coli-based extract preparation for both experienced and new users (Figure 1). Here we provide the step-by-step guide for implementing the CFAI workflow, to go from a streaked plate of cells to a completed CFPS reaction within 24 hours.

Protocol

1. Media growth

1. Prepare 960 mL of CFAI media as described in Table 1 and adjust the pH to 7.2 using KOH.
2. Transfer culture media to a 2.5 L baffled flask and autoclave for 30 min at 121 °C.
3. Prepare a 40 mL sugar solution as described in Table 1. Filter-sterilize the solution into a separate autoclaved glass container.
   NOTE: The sugar solution can be stored in a 30 °C incubator until further use.
4. Allow the media to completely cool to below 40 °C after autoclaving.
5. Prior to inoculation of the CFAI media, add the sugar solution directly to the CFAI media.
6. To inoculate the media, swipe a loopful of colonies from a previously streaked E. coli BL21 Star (DE3) plate and insert directly into the media. Swirl the loop into the media but avoid touching the sides of the container. Ensure that the streak plate is fresh, with viable cells.
7. Place the flask with the inoculated media in a 30 °C incubator while shaking at 200 rpm. Allow the culture to grow overnight. If cells are inoculated in the evening, the culture will reach an approximate optical density, measured at 600 nm (OD₆₀₀), of 10 the subsequent morning. Inoculation and harvest times can be adjusted as needed.

2. Cell harvest

1. Prepare S30 buffer in advance and keep it cold. Prepare the S30 buffer according to Table 2, to a final pH of 8.2.
   NOTE: The S30 buffer can be prepared days prior to the cell harvest. If this is done, prepare without dithiothreitol and store at 4 °C. Add dithiothreitol just before use.
2. From this point forward, keep all solutions and materials on ice. Transfer 1 L of media into a 1 L centrifuge bottle and centrifuge at 5,000 x g between 4-10 °C for 10 min. Decant and dispose of the supernatant. Using a sterile spatula, transfer the pellet to a pre-chilled and previously weighed 50 mL conical tube.
3. Wash once with 30-40 mL of cold S30 buffer by resuspending the pellet via vortexing in 30 s bursts with rest periods on ice.
   NOTE: Due to a higher volume of pellet, splitting the cell pellet into two 50 mL conical tubes can be helpful for the wash step. Storing cells in smaller aliquots also provides flexibility for the downstream processing.
4. Centrifuge the cell resuspension at 5000 x g between 4-10 °C for 10 min.
5. Dispose of the supernatant and wipe any excess from the inside walls of the 50 mL conical tube using a clean tissue, avoid touching the pellet itself. Weigh and flash freeze the pellet in liquid nitrogen. Store at -80 °C until further use.
   ​NOTE: Pellets may not require flash-freezing if the user plans to continue with the extract preparation protocol.

3. Extract preparation

1. Combine the frozen pellet with 1 mL of S30 buffer for every 1 g of the cell pellet and allow to thaw on ice for approximately 30-60 min. Resuspend thawed pellet via vortexing in bursts of 30 s with rest periods on ice. Vortex until there are no visible clumps of cells remaining.
   NOTE: Smaller clumps can be resuspended by mixing using a pipette.
2. Transfer aliquots of 1.4 mL of cell resuspension into 1.5 mL microfuge tubes for cell lysis. Sonicate each tube with a frequency of 20 kHz and 50% amplitude for three bursts of 45 s with 59 s of rest per cycle surrounded by an ice bath. Invert the tube between cycles and immediately add 4.5 µL of 1 M dithiothreitol after the last sonication cycle.
   NOTE: Due to the heat released from sonication, it is extremely important to make sure all aliquots are kept on ice when not being sonicated. The ice bath should be constantly replenished or large enough to stay cool throughout the entire sonication process.
3. Centrifuge each tube at 18,000 x g and 4 °C for 10 min. Retrieve the supernatant and aliquot into 1.5 mL microfuge tubes in 600 µL aliquots. Flash freeze aliquots and store at -80 °C until further use. Care should be taken to pipette only the supernatant.

4. Cell-free protein synthesis

1. Thaw one aliquot of extract from the previous step to perform 15 µL of cell-free protein synthesis reactions in 1.5 mL microfuge tubes in quadruplicate.
2. Prepare each reaction by combining 240 ng of DNA (16 µg/mL final concentration), 2.20 µL of Solution A, 2.10 µL of Solution B, 5.0 µL of extract, and a varying volume of molecular-grade water to fill the reaction to 15 µL. This reaction can be scaled to higher volumes. See Table 3 for ratios.
   1. Prepare Solution A and B according to Table 4. Each solution can be prepared in batches of 100 µL to 1 mL, aliquoted, and stored at -80 °C until further use.
      NOTE: The DNA amount can vary depending on the protein of interest. In this case, the plasmid used, pJL1-sfGFP, has been optimized to perform at 16 µg/mL, or 597 µM.
3. Let the reactions run for at least 4 h at 37 °C.

5. Quantification of reporter protein, super folder green fluorescent protein (sfGFP)

1. Using a half area 96-well black polystyrene plate, combine 2 µL of each cell-free protein synthesis reaction product with 48 µL of 0.05 M HEPES buffer at pH 7.2. Three to four replicates of each reaction tube are recommended.
2. Quantify the fluorescence intensity of the sfGFP with an excitation wavelength of 485 nm and an emission wavelength of 528 nm.
3. For conversion of relative fluorescence units to volumetric yield (µg/mL) of sfGFP, establish a standard curve using purified pJL1-sfGFP.

Representative Results

When preparing CFAI media, glucose was exchanged for an increase in lactose and glycerol as the main energy substrate in the media. Additionally, the buffering capacity of the CFAI media was increased as well. These specific components are given in Table 1.

The cells were then grown to both an OD₆₀₀ of 10 and the standard 2.5 in CFAI media to show consistency with extract quality despite varying extract quantities. The 2.5 OD₆₀₀ CFAI media was grown after inoculating from a seed culture in LB broth at 37 °C, 200 rpm, while the OD₆₀₀ 10 culture was inoculated directly from a plate. Each batch of CFAI media was then monitored and harvested at their respective OD₆₀₀. The growth to an OD₆₀₀ of 10 led to an increase in higher amount of cell pellet and overall extract obtained, as it produced 9.60 mL of extract versus the 2.10 mL of extract obtained from the growth to 2.5 OD₆₀₀ (Figure 2). Further analysis of total protein concentration demonstrated no significant difference in overall protein in each extract (Table 5). Even though they were grown to different levels of optical density, both batches of extract demonstrated similar results in cell-free reactions using sfGFP (Figure 3). This suggests that the combination of the increased buffering capacity, the use of lactose and glycerol as the main carbon source and implementing lactose instead of IPTG for T7RNAP induction help stabilize extract growths to any OD₆₀₀ below 10.

Autoclaved CFAI Media:
Components	Amount
Sodium Chloride	5.0 g
Tryptone	20.0 g
Yeast Extract	5.0 g
Potassium Phosphate, monobasic	6.0 g
Potassium Phosphate, dibasic	14.0 g
NanopureTM Water	Fill to a total of 960 mL
Filter Sterilized Sugar Solution:
Components	Amount
D-Glucose	0.50 g
D-Lactose	4.0 g
80% v/v Glycerol	7.5 mL
NanopureTM Water	28.0 mL

Table 1: CFAI components. Components for CFAI media and sugar solutions with their respective amounts. The media should be stirred throughout the addition of each component and the sugar solution filter sterilized. Each solution should be added to a separate sterile container prior to inoculation.

S30 Buffer
Components	Concentration
Tris Acetate pH 8.2 at room temperature	10 mM
Magnesium Acetate	14 mM
Potassium Acetate	60 mM
Dithiothreitol	2 mM

Table 2: S30 buffer components: Components for S30 buffer were added with their respective amounts into a sterile 50 mL conical tube.

Component	Amount
Solution A	2.20 μL
Solution B	2.1 μL
Extract	5 μL
DNA Template	Volume for 16 μg/mL final
Water	Fill to a total of 15 μL

Table 3: CFPS reaction ratios: Relative volume percentages for Solution A, Solution B, and extract. The DNA volume can vary depending on the specific plasmid's concentration and may need to be optimized for the user's specific plasmid being used.

Solution A		Solution B
Components	Concentration	Components	Concentration
ATP	1.2 mM	Magnesium Glutamate	10 mM
GTP	0.850 mM	Ammonium Glutamate	10 mM
UTP	0.850 mM	Potassium Glutamate	130 mM
CTP	0.850 mM	Phosphoenolpyruvate (PEP)	30 mM
Folinic Acid	31.50 μg/mL	L-Valine	2 mM
tRNA	170.60 μg/mL	L-Tryptophan	2 mM
Nicotinamide Adenine Dinucleotide (NAD)	0.40 mM	L-Isoleucine	2 mM
Coenzyme A	0.27 mM	L-Leucine	2 mM
Oxalic Acid	4.00 mM	L-Cysteine	2 mM
Putrescine	1.00 mM	L-Methionine	2 mM
Spermidine	1.50 mM	L-Alanine	2 mM
HEPES Buffer pH 7.5	57.33 mM	L-Arginine	2 mM
		L-Asparagine	2 mM
		L-Aspartic Acid	2 mM
		L-Glutamic acid	2 mM
		L-Glycine	2 mM
		L-Glutamine	2 mM
		L-Histidine	2 mM
		L-Lysine	2 mM
		L-Proline	2 mM
		L-Serine	2 mM
		L-Threonine	2 mM
		L-Phenylalanine	2 mM
		L-Tyrosine	2 mM

Table 4: Solution A and B components. Stock concentrations for the components for Solution A and B were added with their respective amounts, each in a 1.5 mL microfuge tube.

Extract	Total Protein Concentration (μg/mL)	Standard Deviation
2xYTPG 2.5 OD	30617	3745
CFAI 2.5 OD	30895	2254
CFAI 10.0 OD	27905	3582

Table 5: Total extract protein yields. Analysis of the total protein of different cell extract growths. Total protein concentration was determined using a Bradford Assay. Each concentration was determined from triplicates using a 1:40 dilution.

Figure 1: Comparison of CFAI and typical workflow from cells to CFPS: Comparison of the overall timeline from cells to CFPS using the (A) CFAI workflow (left, red) versus the (B) previously established method (green, right). The comparison demonstrates the reduced researcher oversight and timeline when performing CFPS using the CFAI workflow. Please click here to view a larger version of this figure.

Figure 2: CFAI pellet size comparison. Comparison of CFAI media pellets after cell harvest at different OD₆₀₀. The media grown to an OD₆₀₀ of 2.5 produced a 2.23 g cell pellet (left) and the media grown to an OD₆₀₀ of 10 produced a 9.49 g cell pellet (right). Please click here to view a larger version of this figure.

Figure 3: Effects of growth on CFPS reaction yields. (A) Comparison of CFPS reaction yields between growths to 2.5 OD₆₀₀ and 10 OD_600, with (B) images of each CFPS reaction above their respective yield. Cell-free reactions were performed in a 1.5 mL microfuge tube and quantified after 24 h of incubation at 37 °C using a standard curve to correlate fluorescence to sfGFP concentration. The "Negative" corresponds to the set of negative control reactions in which no template DNA was added. The traditional 2xYTPG media (the positive control) and the CFAI extracts are of similar quality as demonstrated through their high CFPS yields. Please click here to view a larger version of this figure.

Discussion

Researcher oversight is traditionally needed for two key actions during cell growth: the induction of T7 RNAP and harvesting cells at a specific OD₆₀₀. CFAI obviates both of those requirements to decrease the researcher's time and technical training required in order to prepare high quality cell extracts. Auto-induction of T7 RNAP is achieved by replacing glucose with lactose as the primary sugar in the media, obviating the previous need to actively monitor the growth and then induce with IPTG at a precise point during cell growth. The need to actively monitor cell cultures to harvest at a specific OD₆₀₀ is also obviated, untethering the researcher from the cell culture. This adds to the recent work which have also demonstrated production of quality extracts harvested at non-traditional times^13,25,26. The new media formulation improves buffering capacity and carbon sources to support active energy metabolism even as the cell culture approaches stationary phase. The capacity to obtain robust cell extracts from high OD₆₀₀ cultures allows the researcher to harvest the cultures at their convenience²⁷. The workflow we prefer and recommend is to inoculate the culture in the evening and returning to harvest the next morning.

Harvesting cells at a higher OD₆₀₀ also results in a significantly larger quantity of cells obtained for extract preparation. For experienced researchers, it is worth noting that the cell pellet is much darker in color compared to cells grown in 2xYTPG media, even when harvested at an OD₆₀₀ of 2.5. It is also important to note that if the entire cell pellet is being processed at once, the large amount of resuspension obtained per cell pellet when performing lysis via sonication will take some time. Hence, it is important to keep all aliquots cold during this process^11,13,14. The increase in extract volume per growth decreases cost proportionally and supports biomanufacturing applications. With the improvements demonstrated, the CFAI workflow provides an easier protocol for new and experienced users of cell-free technology to produce reproducible, functional E. coli extract.

Despite the advantages of the provided CFAI media, there are limitations to this method. The primary challenge is the nascent nature of the workflow. While metabolomics analysis has shed light on the differences in CFAI OD₆₀₀ 10 extracts as well as reaction products compared to 2xYTPG, the implications of these differences on specific applications remain uncharacterized²⁷. Additionally, this workflow has been developed for BL21 E. coli-based lysate. It is unclear whether the media reformulation would support robust extract preparation from other E. coli strains, such as the genomically recoded strains of E. coli^28,29. It is possible that the CFAI approach could be utilized for generating extracts from other bacterial organisms, but it is unlikely to support extract preparation for eukaryotic organisms such as Chinese Hamster Ovary or rabbit reticulocyte; however, these have their own established methods^30,31. We anticipate that the simplicity of the CFAI workflow will reduce the barriers and incentivize the cell-free community to characterize and evaluate its utility for the broad range of applications that CFPS supports.

Materials

Name	Company	Catalog Number	Comments
1.5 mL Microfuge Tubes	Phenix	MPC-425Q
1L Centrifuge Tube	Beckman Coulter	A99028
Avanti J-E Centrifuge	Beckman Coulter	369001
CoA	Sigma-Aldrich	C3144-25MG
Cytation 5 Cell Imaging Multi-Mode Reader	Biotek	BTCYT5F
D-Glucose	Fisher	D16-3
D-Lactose	Alfa Aesar	J66376
DTT	ThermoFisher	15508013
Folinic Acid	Sigma-Aldrich	F7878-100MG
Glycerol	Fisher	BP229-1
Glycine	Sigma-Aldrich	G7126-100G
HEPES	ThermoFisher	11344041
IPTG	Sigma-Aldrich	I6758-1G
JLA-8.1000 Rotor	Beckman Coulter	366754
K(Glu)	Sigma-Aldrich	G1501-500G
K(OAc)	Sigma-Aldrich	P1190-1KG
KOH	Sigma-Aldrich	P5958-500G
L-Alanine	Sigma-Aldrich	A7627-100G
L-Arginine	Sigma-Aldrich	A8094-25G
L-Asparagine	Sigma-Aldrich	A0884-25G
L-Aspartic Acid	Sigma-Aldrich	A7219-100G
L-Cysteine	Sigma-Aldrich	C7352-25G
L-Glutamic Acid	Sigma-Aldrich	G1501-500G
L-Glutamine	Sigma-Aldrich	G3126-250G
L-Histadine	Sigma-Aldrich	H8000-25G
L-Isoleucine	Sigma-Aldrich	I2752-25G
L-Leucine	Sigma-Aldrich	L8000-25G
L-Lysine	Sigma-Aldrich	L5501-25G
L-Methionine	Sigma-Aldrich	M9625-25G
L-Phenylalanine	Sigma-Aldrich	P2126-100G
L-Proline	Sigma-Aldrich	P0380-100G
L-Serine	Sigma-Aldrich	S4500-100G
L-Threonine	Sigma-Aldrich	T8625-25G
L-Tryptophan	Sigma-Aldrich	T0254-25G
L-Tyrosine	Sigma-Aldrich	T3754-100G
Luria Broth	ThermoFisher	12795027
L-Valine	Sigma-Aldrich	V0500-25G
Mg(Glu)2	Sigma-Aldrich	49605-250G
Mg(OAc)2	Sigma-Aldrich	M5661-250G
Microfuge 20	Beckman Coulter	B30134
Molecular Grade Water	Sigma-Aldrich	7732-18-5
NaCl	Alfa Aesar	A12313
NAD	Sigma-Aldrich	N8535-15VL
New Brunswick Innova 42/42R Incubator	Eppendorf	M1335-0000
NH4(Glu)	Sigma-Aldrich	09689-250G
NTPs	ThermoFisher	R0481
Oxalic Acid	Sigma-Aldrich	P0963-100G
PEP	Sigma-Aldrich	860077-250MG
Potassium Phosphate Dibasic	Acros, Organics	A0382124
Potassium Phosphate Monobasic	Acros, Organics	A0379904
PureLink HiPure Plasmid Prep Kit	ThermoFisher	K210007
Putrescine	Sigma-Aldrich	D13208-25G
Spermidine	Sigma-Aldrich	S0266-5G
Tris(OAc)	Sigma-Aldrich	T6066-500G
tRNA	Sigma-Aldrich	10109541001
Tryptone	Fisher Bioreagents	73049-73-7
Tunair 2.5L Baffled Shake Flask	Sigma-Aldrich	Z710822
Ultrasonic Processor	QSonica	Q125-230V/50HZ
Yeast Extract	Fisher Bioreagents	1/2/8013