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.
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.
The use of cell-free protein synthesis (CFPS) for biotechnology applications has grown substantially over the past few years1,2,3. This development can be attributed in part to increased efforts in understanding the processes that occur in CFPS and the role of each component4,5. Additionally, reduced costs attributed to optimized set-ups and alternative energy sources have made cell-free technology easier to implement for new users6,7,8,9. 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 alike1,11,12,13,14. Cell extract is usually obtained through the lysis of a cell culture, which can be grown using different organisms depending on the specific use desired1,15,16.
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 mRNA4,14,18,19. Under the lacUV5 promoter, isopropyl-thiogalactopyranoside (IPTG) induction allows the expression of T7 RNAP20,21. 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 cavitation1,11,12,22.
The process of bacterial culture and harvesting is consistent across most platforms when using E. coli, but requires multiple days and intense researcher oversight1,11,13. 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 2.514,20. 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 phase23,24. 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.
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 pr…
The authors have nothing to disclose.
Authors would like to acknowledge Dr. Jennifer VanderKelen and Andrea Laubscher for technical support. Authors would also like to thank Nicole Gregorio, Max Levine, Alissa Mullin, Byungcheol So, August Brookwell, Elizabeth (Lizzy) Vojvoda, Logan Burrington and Jillian Kasman for helpful discussions. Authors also acknowledge funding support from the Bill and Linda Frost Fund, Center for Applications in Biotechnology's Chevron Biotechnology Applied Research Endowment Grant, Cal Poly Research, Scholarly, and the National Science Foundation (NSF-1708919).
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 |