Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.
The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 μL batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of > 260 μg/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1−2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.
Cell-free protein synthesis is a versatile and cost-effective method for the expression of valuable proteins or peptides1,2,3,4. Historically, cell-free protein synthesis has been performed using Escherichia coli expression systems; however, there has been a recent surge in using alternative, non-model organisms with novel properties as chassis for cell-free expression5,6,7,8,9,10,11,12. Organisms with unique metabolic profiles are prime candidates as alternatives to E. coli cell-free systems. For example, the marine bacterium Vibrio natriegens is the fastest growing of all known organisms with an observed doubling time of less than 10 min13. This has garnered V. natriegens considerable attention as an emerging microbial host for research and biotechnology14,15,16,17,18. Given that the rapid growth rate of V. natriegens has been linked to high rates of protein synthesis and metabolic efficiency19,20,21, harnessing its cellular machinery for cell-free synthesis may significantly expand the toolkit for rapid protein production and high-throughput screening.
Recently, a cell-free V. natriegens expression system has been demonstrated which is capable of producing super folder GFP (sfGFP) at concentrations of > 260 μg/mL in 3 h with a T7 promoter9. The overall aim of developing this method was to provide users with a highly accessible, cost-efficient, reproducible, and high-yielding cell-free protein expression system that can be prepared using common lab equipment in a short amount of time. This protocol utilizes 1 L cultures in shake flasks, cell lysis by pulse sonication, and small-scale batch reactions in a 96- or 384-well format to maximize parallelization and screening throughput. A long, sustained protein expression is made possible by supplementation of 3-phosphoglyceric acid (3-PGA) as an energy source8,22,23. Upon successfully completing this protocol, a user will have the capability to express a desired protein or set of proteins in a cell-free format using V. natriegens crude cell extract.
Starting from a glycerol stock, V. natriegens crude cell extracts are prepared from cells harvested at an optical density at 600 nm (OD600) of 1.0. A 1 L culture will yield approximately 2−3 mL of extract, which is sufficient for more than 800 cell-free reactions at 25% crude cell extract. Proteins can be expressed using plasmid DNA, linear DNA, or mRNA template; however, linear DNA template degradation by endogenous nucleases remains a major drawback when using wild type V. natriegens cell-free system9. Starting from V. natriegens cultures, protein useable for downstream applications can be achieved by a single user in 1−2 full days.
1. Preparation of V. natriegens Crude Cell Extracts – Bacterial Culture
2. Preparation of V. natriegens Crude Cell Extracts – Cell Lysis
3. Preparation of Cell-free Reaction Components
4. Performing Cell-free Protein Eexpression Reactions Using V. natriegens Crude Extract
5. Calibration of V. natriegens Cell-free Reactions with sfGFP
NOTE: This section is optional. The optimal cell-free reaction ion concentration can vary slightly for each crude extract preparation based on the conditions used for cell lysis. Consider performing the optional cell-free reaction ion calibration protocol described below using sfGFP if reaction yields are significantly lower than expected (concentrations < 1.0 μg/mL).
The described protocol for protein production using V. natriegens cell-free expression system can be executed in 1−2 days by a single user, starting from inoculation of culture to protein available for downstream applications. Preparation of crude cell extracts and master mix stocks comprise a significant portion of this time; however, once prepared, most bulk reagents can be stored long term (Table 7) and used as needed, shortening the time needed to complete the protocol.
The expression system described is best used with plasmid DNA template or mRNA generated by in vitro transcription reactions. While linear DNA can also be used as template for protein production, it yields significantly lower amount of protein. For example, at optimal reactions conditions at 26 °C, a single 10 μL cell-free reaction can produce > 260 μg/mL of sfGFP in 3 hours using 0.3 pmol of plasmid DNA template or a comparable > 125 μg/mL of sfGFP using 14 pmol of mRNA transcript (Figure 5A,B). However, 0.3 pmol of linear DNA will produce significantly less protein (< 20 μg/mL). For each template type, the majority of the protein will be produced within 1−1.5 h; however, it is recommended that reaction is run for a minimum of 3 hours. Cell-free reaction concentrations of sfGFP were determined via linear regression using a purified sfGFP standard curve measuring fluorescence at Ex/Em = 485 nm/528 nm.
The yield of protein production is significantly affected by the concentration of potassium and magnesium ions (K+ and Mg2+, respectively). Under optimized conditions, it was found that the optimal Mg2+ and K+ ion concentrations will be 3.5 mM and 80 mM, respectively (Figure 6A,B). Deviations from the optimal ion concentrations may result in a decreased capacity for the cell-free expression system to produce protein with appreciable yields. Additional calibration may be necessary if reaction yields are significantly lower than expected. An optional protocol for ion calibration is described in section 5. This allows for some flexibility in compensating for crude cell extract variability incurred from individual cell lysis equipment and conditions.
Figure 1: A close-up view of the beaker and tube holder on the sonication platform. Please click here to view a larger version of this figure.
Figure 2: Sonication equipment setup for the preparation of crude cell extract. Please click here to view a larger version of this figure.
Figure 3: A view of the pellet after sonication and centrifugation. Please click here to view a larger version of this figure.
Figure 4: Flash freezing dip for extract storage. Please click here to view a larger version of this figure.
Figure 5: Representative results for V. natriegens cell-free protein synthesis using different template types. (A) endpoint assay of sfGFP production using equimolar concentrations of plasmid and linear DNA template (0.3 pmol) as well as increasing concentrations of mRNA template. Cell-free sfGFP concentration was determined using a standard curve of purified sfGFP measured at Ex/Em = 485 nm/528 nm after 180 minutes of incubation at 26 °C at optimal reaction conditions in 10 μL volumes. (B) Kinetic assay of sfGFP production using equimolar concentrations of plasmid and linear DNA template as well as increasing concentrations of mRNA template. sfGFP measurements were taken every 3 minutes at Ex/EM = 485 nm/528 nm over 180 minutes of total incubation time at optimal reaction conditions in 10 μL volumes. For both endpoint and kinetic assays, samples were blank corrected using cell-free reactions supplemented with all components except template. The mean and standard deviations are shown (n = 3). Please click here to view a larger version of this figure.
Figure 6: Representative results for ion concentration calibration. (A) V. natriegens cell-free reactions were supplemented with increasing concentrations of Mg2+ and incubated at 26 °C for 180 minutes in 10 μL reactions. The total concentration of K+ was 160 mM for all Mg2+ concentrations. Cell-free sfGFP concentration was determined using a standard curve of purified sfGFP measured at Ex/Em = 485 nm/528 nm. (B) V. natriegens cell-free reactions were supplemented with increasing concentrations of K+ and incubated at 26 °C for 180 min in 10 μL reactions. The total concentration of Mg2+ was 3.5 mM for all K+ concentrations. For both calibrations, samples were blank corrected using cell-free reactions supplemented with all components except template. The mean and standard deviations are shown (n = 3). This figure has been modified from Wiegand et al.9. Reprinted with permission from Wiegand, D.J., Lee, H.H., Ostrov, N., Church, G.M. Establishing a Cell-Free Vibrio natriegens Expression System. ACS Synthetic Biology. 7 (10), 2475−2479 (2018). Copyright 2018 American Chemical Society. Please click here to view a larger version of this figure.
Table 1 A | Preparation of LB-V2 Bacterial Growth Media | ||
Component | Quantity (g) | Final Concentration (mM) | Final Volume (L) |
LB Broth (Miller) | 25 | 1 | |
NaCl | 11.69 | 200 | |
MgCl2 | 2.20 | 23.1 | |
KCl | 0.31 | 4.2 | |
Table 1 B | Preparation of S30A Lysis Buffer | ||
Component | Quantity (g) | Final Concentration (mM) | Final Volume (L) |
Tris Solution (pH 8.0) - 1 M (mL)* | 25 | 50 | 0.5 |
Mg-glutamate | 2.72 | 14 | |
K-glutamate | 6.10 | 60 | |
Dithiothreitol (DTT) - 1 M (mL)* | 1 | 2 |
Table 1: Reagents for the preparation of 1 L of LB-V2 bacterial growth media and 0.5 L of S30A cell lysis buffer. Please click here to download this file in Excel.
Preparation of 10x Energy Solution Master Mix | ||||
Component | Stock Concentration (mM) | Final Concentration (mM) | Quantity (µL) | Final Volume (µL) |
HEPES-KOH pH 8 | 1750 | 500 | 1428.57 | 5000 |
ATP | 100 | 15 | 750.00 | |
GTP | 100 | 15 | 750.00 | |
CTP | 100 | 9 | 450.00 | |
UTP | 100 | 9 | 450.00 | |
tRNA from E. coli MRE 600 (mg/mL)* | 100 | 2 | 100.00 | |
Coenzyme A Hydrate | 200 | 2.6 | 65.00 | |
NAD | 200 | 3.3 | 82.50 | |
cAMP | 650 | 7.5 | 57.69 | |
Folinic Acid | 100 | 0.7 | 35.00 | |
Spermidine | 1600 | 10 | 31.25 | |
3-PGA | 2000 | 300 | 750.00 | |
Sterile Deionized Water | 49.99 | |||
Preparation of 4x Amino Acid Master Mix | ||||
Component | Stock Concentration (mM) | Final Concentration (mM) | Quantity (µL) | Final Volume (µL) |
ALA | 168 | 8 | 114.3 | 2400 |
ARG | 168 | 8 | 114.3 | |
ASN | 168 | 8 | 114.3 | |
ASP | 168 | 8 | 114.3 | |
GLN | 168 | 8 | 114.3 | |
GLU | 168 | 8 | 114.3 | |
GLY | 168 | 8 | 114.3 | |
HIS | 168 | 8 | 114.3 | |
IIE | 168 | 8 | 114.3 | |
LYS | 168 | 8 | 114.3 | |
MET | 168 | 8 | 114.3 | |
PHE | 168 | 8 | 114.3 | |
PRO | 168 | 8 | 114.3 | |
SER | 168 | 8 | 114.3 | |
THR | 168 | 8 | 114.3 | |
VAL | 168 | 8 | 114.3 | |
TRP | 168 | 8 | 114.3 | |
TYR | 168 | 8 | 114.3 | |
LEU | 140 | 8 | 137.1 | |
CYS | 168 | 8 | 114.3 | |
Sterile Deionized Water | 91.4 |
Table 2: Reagents for the preparation of 5 mL of 10x energy solution master mix and 2.4 mL of 4x amino acid master mix. Please click here to download this file in Excel.
T7 RNA Polymerase In Vitro Transcription Reactions | ||||
Component | Stock (mM) | Final Concentration (mM) | Quantity (µL) 1x Reaction | Quantity (µL) 50x Reactions |
10x RNAPol Reaction Buffer | 1.00 | 50 | ||
ATP | 100 | 0.5 | 0.10 | 5 |
GTP | 100 | 0.5 | 0.10 | 5 |
CTP | 100 | 0.5 | 0.10 | 5 |
UTP | 100 | 0.5 | 0.10 | 5 |
DNA Template (ng/µL)* | 1000 | 500 | 0.50 | 25 |
T7 RNA Polymerase | 200 | 2.6 | 2.00 | 100 |
Rnase Inhibitor, Murine | 200 | 3.3 | 0.50 | 25 |
Sterile Deionized Water | 15.60 | 780 | ||
Reaction Volume (µL): | 20 |
Table 3: In vitro transcription components for mRNA generation. Please click here to download this file in Excel.
Cell-free Reaction Master Mix | ||||
Component | Stock Concentration (mM) | Final Concentration (mM) | Quantity (µL) 1x Reaction | Quantity (µL) 50x Reactions |
Extract (%)* | 25 | 2.50 | 125.00 | |
Mg-glutamate | 100 | 3.5 | 0.35 | 17.50 |
K-glutamate | 2000 | 80 | 0.40 | 20.00 |
4x Amino Acid Master Mix | 8.0 | 2 | 2.50 | 125.00 |
10x Energy Solution Master Mix | 1.00 | 50.00 | ||
Plasmid DNA (ng/µL)* | 1000 | 500 | 0.50 | 25.00 |
50% PEG-8000 (%)* | 50 | 2 | 0.40 | 20.00 |
T7 RNA Polymerase | 1.00 | 50.00 | ||
RNase Inhibitor, Murine | 0.10 | 5.00 | ||
Sterile Deionized Water | 1.25 | 62.50 | ||
Reaction Volume (µL): | 10 | |||
Alternative Cell-free Reaction Master Mix for mRNA Template | ||||
Component | Stock Concentration (mM) | Final Concentration (mM) | Quantity (µL) 1x Reaction | Quantity (µL) 50x Reactions |
Extract (%)* | 25 | 2.50 | 125.00 | |
Mg-glutamate | 100 | 3.5 | 0.35 | 17.50 |
K-glutamate | 2000 | 80 | 0.40 | 20.00 |
4x Amino Acid Master Mix | 8.0 | 2 | 2.50 | 125.00 |
10x Energy Solution Master Mix | 1.00 | 50.00 | ||
mRNA Template (ng/µL)* | 2000 | 4000 | 2.00 | 100.00 |
50% PEG-8000 (%)* | 50 | 2 | 0.40 | 20.00 |
RNase Inhibitor, Murine | 0.10 | 5.00 | ||
Sterile Deionized Water | 0.75 | 37.50 | ||
Reaction Volume (µL): | 10 |
Table 4: Components of optimized V. natriegens cell-free reaction master mix for DNA template and mRNA template. Please click here to download this file in Excel.
Mg2+ Calibration | Quantity (µL) 1x Reaction | Quantity (µL) 1x Reaction | Quantity (µL) 100x Reactions | Quantity (µL) 100x Reactions | |
Final (mM) | Stock | 100 mM Mg-Glu | diH20 | 100 mM Mg-Glu | Deionized H2O |
2.5 | 0 | 0.00 | 1.00 | 0 | 100 |
3.5 | 1 | 0.10 | 0.90 | 10 | 90 |
4.5 | 2 | 0.20 | 0.80 | 20 | 80 |
5.5 | 3 | 0.30 | 0.70 | 30 | 70 |
Reaction Volume (µL): | 10 | ||||
K+ Calibration | Quantity (µL) 1x Reaction | Quantity (µL) 1x Reaction | Quantity (µL) 100x Reactions | Quantity (µL) 100x Reactions | |
Final (mM) | Stock | 2000 mM K-Glu | diH20 | 2000 mM K-Glu | Deionized H2O |
40 | 30 | 0.15 | 1.85 | 12 | 148 |
80 | 70 | 0.35 | 1.65 | 28 | 132 |
160 | 150 | 0.75 | 1.25 | 60 | 100 |
320 | 310 | 1.55 | 0.45 | 124 | 36 |
Reaction Volume (µL): | 10 | ||||
Ion Calibration Cell-free Reaction Master Mix | |||||
Component | Stock Concentration (mM) | Final Concentration (mM) | Quantity (µL) 1x Reaction | Quantity (µL) 50x Reactions | |
Extract (%)* | 25 | 2.50 | 125.00 | ||
Mg-glutamate | Variable | Variable | 1.00 | 50.00 | |
K-glutamate | Variable | Variable | 2.00 | 100.00 | |
4x Amino Acid Master Mix | 8.0 | 2 | 2.50 | 125.00 | |
10x Energy Solution Master Mix | 1.00 | 50.00 | |||
Plasmid DNA (ng/µL)* | 1000 | 250 | 0.25 | 12.50 | |
50% PEG-8000 (%)* | 50 | 2 | 0.40 | 20.00 | |
T7 RNA Polymerase | 1.00 | 50.00 | |||
RNase Inhibitor, Murine | 0.10 | 5.00 | |||
Sterile Deionized Water | 0.00 | 0.00 | |||
Reaction Volume (µL): | 10 |
Table 5: Ion concentration calibration master mixes. Please click here to download this file in Excel.
CF Ion Calibration Map | 40 mM K+ | 80 mM K+ | 160 mM K+ | 320 mM K+ | |||||||||
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | ||
2.5 mM Mg2+ | A | ||||||||||||
3.5 mM Mg2+ | B | ||||||||||||
4.5 mM Mg2+ | C | ||||||||||||
5.5 mM Mg2+ | D |
Table 6: Ion calibration map. Please click here to download this file in Excel.
Storage Conditions and Shelf Lives of CF Components | ||
Component | Storage Location | Shelf Life |
Sterile LB-V2 Growth Media | 4 °C | 3-6 months |
V. natriegens Crude Cell Extract | -80 °C | 1-3 weeks |
100 mM Mg-Glutamate | Room Temp | 6 months |
2000 mM K-Glutamate | Room Temp | 6 months |
50% PEG-8000 | Room Temp | 6 months |
10x Energy Solution Master Mix | -80 °C | 3-6 months |
4x Amino Acid Master Mix | -80 °C | 3-6 months |
Plasmid/Linear DNA Template | -20 °C | 6-12 months |
mRNA Template | -80 °C | 3-4 weeks |
Table 7: Cell-free storage conditions and shelf lives. Please click here to download this file in Excel.
This protocol has been optimized for wild-type V. natriegens and bacterial growth media comprised of LB supplemented with V2 salts (Table of Materials). Other strains of V. natriegens can be similarly cultured to generate crude cell extracts for cell-free reactions; however, their use requires additional optimization of this protocol. Additionally, this cell-free protein expression system has been optimized using 3-phosphoglyceric acid (3-PGA) as the primary energy regeneration source. Other energy regeneration sources may be used; however, optimization of reagents and calibration will likely be required to obtain high yielding protein expression10,11.
Specific attention to several critical steps in this protocol will ensure maximal extract productivity for high-yielding cell-free protein production. First, crude cell extract must be prepared from V. natriegens cell cultures harvested in a mid-exponential phase of growth; protein yield is maximal when cultures reach an OD600 = 1.0 ± 0.2. While cell-free protein production is possible from cells harvested at a range of optical densities, we have previously found that cells harvested in an exponential state of growth yield significantly more protein9. The observed effects of optical density on crude cell extract performance are consistent with those reported for other cell-free expression systems derived from cells grown in batch culture conditions1. Because V. natriegens grows at a rapid rate, it is critical to closely monitor cultures’ optical densities. In general, it is expected that V. natriegens cultures should reproducibly reach an OD600 of 1.0 within 1−1.5 h using this protocol; however, individual growth conditions such as the use of baffled versus non-baffled flasks, which affects aeration, or air versus water incubation, which affects the rate and stability of incubation temperature, may alter growth time. Furthermore, it is generally recommended to culture at least 250 mL of V. natriegens in a 1 L baffled flask to ensure a large cell pellet at harvest for easy manipulation and transfer. This greatly improves the success of crude cell extract preparation as well as increases the total volume of extract produced from a pellet. When using smaller scale preparation, culture conditions and reagents can be adjusted appropriately. For large-scale fermentation, further optimization of culture conditions may be required. Finally, to ensure high protein yield, it is critical that cell pellets are processed immediately after harvesting, or within 1−2 days of storing at -80 °C.
The proper lysis of the cell pellet by pulse sonication is critical to the success of cell-free protein expression and is often the most difficult aspect of this protocol for new users. Typically, a well lysed pellet will yield a significant volume of liquid extract that is free of debris. The extract should be slightly viscous but can be easily pipetted when aliquoting into storage tubes before flash freezing in liquid nitrogen. Figure 3 depicts a representation of a well lysed pellet (Figure 3A) in comparison to a poorly lysed pellet (Figure 3B) after the post lysis centrifugation step. A major indication of complete cell lysis is a crude cell extract total protein concentration > 20 mg/mL as determined by a total protein assay (step 2.13). Over-sonication or excessive heating of the crude cell extract will damage the cellular machinery, which cannot be determined without performing a cell-free reaction. Thus, it is highly beneficial to test extract efficiency with a control reaction before dedicating significant time and effort to downstream protein expression applications. While additional optimization may be necessary for different sonication equipment, the pulse sonication steps described have been highly reproducible in our hands.
The use of linear DNA template derived from PCR amplification, restriction enzyme digest, or commercial gene synthesis can significantly increase the capacity for high-throughput and rapid protein production in cell-free expression systems24. While protein production from PCR amplified linear template has been demonstrated, the yield of these reactions are approximately 13.5-fold lower compared to reactions using plasmid DNA template at equimolar ratios9. This is primarily due to the instability of the linear DNA template which is likely degraded by endogenous nucleases present in V. natriegens crude cell extract. While lambda phage protein GamS has been previously used to protect Linear DNA template24,25, it was found to be incompatible with V. natriegens extracts9. Additionally, while increasing the concentration of linear DNA template may allow for a higher protein yield, its fast degradation in crude cell extract will still be a major problem.
A solution to overcoming linear DNA template degradation may be to supplement cell-free reactions with mRNA template generated from in vitro transcription of linear DNA. On the other hand, the use of an RNase inhibitor offers significant protection against mRNA transcript degradation and appreciable protein yields can be obtained in the 10 μL cell-free reaction format (Figure 5A,B). Cloning of linear DNA into a circular template through TA ligation, TOPO cloning, Golden Gate assembly, or other recombination methods may be used to circumvent template degradation. Nevertheless, further approaches for inhibition of nuclease activity will be necessary for efficient protein expression using linear DNA template.
To date, several different approaches have been proposed for preparation of crude extract for cell-free protein expression9,10,11,26. In developing this protocol, we sought to maximize user accessibility, reduce overall cost, and minimize time-consuming steps. For example, a high protein yield is achieved using a simple two-step sonication-centrifugation process, and does not require cell homogenizers, lengthy dialysis steps or run-off reaction. It is simple to execute in a short period of time and does not require high level of laboratory expertise. Thus, it can help facilitate cell-free expression as a standard for translational academic research and industrial process design.
This protocol expands the toolkit available for investigation and utility of V. natriegens, a non-model organism with unique biological properties. Higher protein yield can be achieved by employing semi- or fully-continuous cell-free reactions, to allow for energy regeneration, resupplying of amino acids, and the removal of waste products3,5,27. Furthermore, the engineering of wild-type V. natriegens to produce DNAse- or RNAse-deficient strains, removal of deleterious and competing metabolic pathways, and expression of additional tRNAs could greatly enhance the production of proteins in this system28,29. As we unravel the biology underlying its rapid growth, further development of V. natriegens cell-free systems may accelerate bioproduction capabilities and enable robust expression of therapeutic peptides, small molecules, and synthetic materials.
This work was funded by the National Institute of General Medical Sciences 1U01GM110714-01 and Department of Energy DE-FG02-02ER63445. The authors would like to thank Dr. Richard Kohman, Dr. Jenny Tam, and Dr. Edgar Goluch for helpful advice on constructing the protocol section of this manuscript.
Name | Company | Catalog Number | Comments |
15 mL Tubes | Corning | 352196 | |
2 mL Tubes | Eppendorf | 22363352 | |
384-well Black Assay Plates | Corning | 3544 | |
384-well PCR Plates | Eppendorf | 951020702 | |
50 mL Tubes | Corning | 352070 | |
96-well PCR Plates | Eppendorf | 30129300 | |
Adenosine 3',5'-cyclic monophosphate sodium salt monohydrate | Sigma | A6885 | |
Adenosine 5'-triphosphate - 100 mM | NEB | N0450L | |
Applied Biosystems Veriti 384-well Thermocycler | ThermoFisher | 4388444 | |
Assay Plate Adhesives | BioRad | MSB1001 | |
β-Nicotinamide adenine dinucleotide hydrate (NAD) | Sigma/Roche | 10127965001 | |
Coenzyme A hydrate | Sigma | C4283 | |
Cytidine 5'-triphosphate - 100 mM | NEB | N0450L | |
D-(-)-3-Phosphoglyceric acid disodium salt (3-PGA) | Sigma | P8877 | |
Dewar Flask - 4L | ThermoScientific | 10-194-100C | |
DL-Dithiothreitol solution - 1 M | Sigma | 42816 | |
Folinic acid calcium salt hydrate | Sigma | 47612 | |
Glacial Acetic Acid | Sigma | A6283 | |
Guanosine 5'-triphosphate - 100 mM | NEB | N0450L | |
HEPES | Sigma | H3375 | |
L-Glutamic acid hemimagnesium salt tetrahydrate | Sigma | 49605 | |
L-Glutamic acid potassium salt monohydrate | Sigma | G1149 | |
LB Broth (Miller) | Sigma | L3522 | |
Magnesium chloride (MgCl2) | Sigma | M8266 | |
Plasmid pJL1-sfGFP | Addgene | 69496 | |
Plasmid Plus Maxi kit | Qiagen | 12963 | |
Poly(ethylene glycol) (PEG)-8000 | Sigma | 89510 | |
Potassium chloride (KCl) | Sigma | P9333 | |
Potassium hydroxide Pellets | Sigma/Roche | 1050121000 | |
Q125 Sonicator and CL-18 probe with a ⅛-inch tip | Qsonica | 4422 | |
RNA Clean and Concentrator Kit | Zymo | R1013 | |
RNase Inhibitor, Murine | NEB | M0314 | |
RTS Amino Acid Sampler | Biotechrabbit | BR1401801 | |
Sodium chloride (NaCl) | Sigma | S7653 | |
Spermidine | Sigma | S0266 | |
T7 RNA Polymerase | NEB | M0251 | |
Tris Solution (pH 8.0) - 1 M | Invitrogen | AM9856 | |
tRNA from E. coli MRE 600 | Sigma/Roche | 10109541001 | |
Uridine 5'-triphosphate - 100 mM | NEB | N0450L | |
Vibrio natriegens (Wild-type) Lyophilized Stock | ATCC | 14048 |
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