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* These authors contributed equally
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.
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.
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 years1,2. 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 cell3,4,5,
1. Media Preparation and Cell Growth
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.......
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.......
Authors would like to acknowledge Dr. Jennifer VanderKelen, Andrea Laubscher, and Tony Turretto for technical support, Wesley Kao, Layne Williams, and Christopher Hight 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 Creative Activities Grant Program (RSCA 2017), and the National Science Foundation (NSF-1708919). MZL acknowledges the California State University Graduate Grant. MCJ acknowledges the Army Research Office W911NF-16-1-0372, National Science Foundation grants MCB-141356....
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 |
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