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Method Article
* These authors contributed equally
Presented here is a protocol for the preparation and buffer calibration of cell extracts from exonuclease V knockout strains of Escherichia coli BL21 Rosetta2 (ΔrecBCD and ΔrecB). This is a fast, easy, and direct approach for expression in cell-free protein synthesis systems using linear DNA templates.
Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA templates for CFPS will further enable the technology to reach its full potential, decreasing the experimental time by eliminating the steps of cloning, transformation, and plasmid extraction. Linear DNA can be rapidly and easily amplified by PCR to obtain high concentrations of the template, avoiding potential in vivo expression toxicity. However, linear DNA templates are rapidly degraded by exonucleases that are naturally present in the cell extracts. There are several strategies that have been proposed to tackle this problem, such as adding nuclease inhibitors or chemical modification of linear DNA ends for protection. All these strategies cost extra time and resources and are yet to obtain near-plasmid levels of protein expression. A detailed protocol for an alternative strategy is presented here for using linear DNA templates for CFPS. By using cell extracts from exonuclease-deficient knockout cells, linear DNA templates remain intact without requiring any end-modifications. We present the preparation steps of cell lysate from Escherichia coli BL21 Rosetta2 ΔrecBCD strain by sonication lysis and buffer calibration for Mg-glutamate (Mg-glu) and K-glutamate (K-glu) specifically for linear DNA. This method is able to achieve protein expression levels comparable to that from plasmid DNA in E. coli CFPS.
Cell-free protein synthesis (CFPS) systems are increasingly being used as a fast, simple, and efficient method for biosensor engineering, decentralized manufacturing, and prototyping of genetic circuits1. Due to their great potential, CFPS systems are used regularly in the field of synthetic biology. However, so far CFPS systems rely on circular plasmids that can limit the technology from reaching its full potential. Preparing plasmid DNA depends on many time-consuming steps during cloning and large amounts of DNA isolation. On the other hand, PCR amplification from a plasmid, or a synthesized DNA template, can be used to simply prepare CFPS templates within a few hours2,3. Therefore, application of linear DNA offers a promising solution for CFPS. However, linear DNA is rapidly degraded by exonucleases naturally present in cellular extracts4. There are solutions that address this problem, such as using the λ-phage GamS protein5 or DNA containing Chi sites6 as protective agents, or directly protecting the linear DNA by chemical modification of its ends2,7,8,9. All these methods require supplementations to the cell extract, which are costly and time-consuming. It has been known for a long time that the exonuclease V complex (RecBCD) degrades linear DNA in cell lysates4. Recently, we showed that linear DNA can be much better protected in lysates from cells knocked out for exonuclease genes (recBCD)10.
In this protocol, steps for the preparation of cell-free lysates from the E. coli BL21 Rosetta2 ΔrecBCD strain by sonication lysis are described in detail. Sonication lysis is a common and affordable technique employed by several labs11,12. The extracts produced from this strain do not need the addition of any extra component or DNA template modification to support expression from linear DNA templates. The method relies on the essential step of buffer optimization for cell extracts specifically for linear DNA expression from native E. coli promoters. It has been shown that this specific buffer optimization for linear DNA expression is the key for native σ70 promoters to yield high protein production without GamS protein or Chi DNA supplementation, even avoiding purification of the PCR products10. The optimal concentration of Mg-glu for linear DNA expression was found to be similar to that for plasmid DNA. However, the optimal concentration of K-glu showed a substantial difference between linear and plasmid DNA, likely due to a transcription-related mechanism10. The functionality of proteins expressed using this method has been demonstrated for several applications, such as rapid screening of toehold switches and activity assessment of enzyme variants10.
This protocol provides a simple, efficient, and cost-effective solution for using linear DNA templates in E. coli cell-free systems by simply using mutant ΔrecBCD cell extracts and specific calibration for linear DNA as template.
1. Media and buffer preparation
2. Cell culture and lysate preparation (4 day experiment)
3. Cell-free buffer calibration for linear DNA
NOTE: Cell-free buffer was calibrated for optimal Mg-glu and K-glu concentrations as described in Sun et al.13. Supplementary File 1 is needed for the calibration steps. Reactions were set to a final volume of 10.5 µL each. Extracts were calibrated using 1 nM of linear (see section 4 below) or plasmid DNA. The experiments can be performed on the same day the buffers are prepared, or the prepared buffers can be frozen at -80 °C to perform the experiment on another day. For all calibration steps, each component was thawed on ice before mixing and pipetting into a 384-well plate.
4. Linear DNA preparation
NOTE: For lysate calibration, plasmid P70a-deGFP is used. This plasmid was maintained in E. coli KL740 cl857+ (Table 6) for miniprep using the Plasmid Miniprep Kit, or maxiprep using the Plasmid Maxiprep Kit. Linear DNA fragments used as expression templates in the cell-free reactions were PCR amplified using the primers and templates listed in Table 7 and Table 8.
5. Experimental execution
NOTE: In addition to using Supplementary File 1 to calibrate the buffer stock for each lysate batch prepared (above), it is recommended to use the ‘Reaction Preparation’ tab in the file to set up the subsequent cell-free reactions. As before, the reactions' volume is set at 10.5 µL per reaction, of which only 10 µL is finally pipetted into the 384-well plate for data acquisition. Here, 5 nM of each template is used for cell-free expression. It is important to be consistent when pipetting the reactions-use the same pipette and the type of tips. Dispense carefully to avoid any bubbles in the well or any liquid sticking to the walls of the well. If needed, spin down the plate (<2,500 x g, 1 min, room temperature).
6. FITC and GFP relative quantification
NOTE: GFP expression is exported by the plate reader in arbitrary fluorescence units (a.u.). However, it is recommended to use standardized units of measurement in order to compare fluorescence values between different settings (batches, equipment, users, and laboratories). Presented here are detailed steps to convert the fluorescence values (a.u.) to FITC-equivalent and eGFP (µM) values, using standard curves of NIST-FITC and recombinant eGFP. Store NIST-FITC stock solution at 4 °C and store recombinant eGFP at -20 °C. Ensure that the stock solution and serial dilutions are protected from light. Upon delivery, it is recommended to aliquot the recombinant eGFP into smaller volumes to avoid multiple freeze-thaw cycles.
Representative results are shown here after calibration of the lysate for optimal Mg-glutamate and K-glutamate levels separately for linear and plasmid DNA (Figure 1). The Mg-glutamate optimal concentration is similar across ΔrecB and ΔrecBCD extracts at 8 mM (Figure 1B). However, the optimal K-glutamate concentration for plasmid DNA is 140 mM, whereas the optimal K-glutamate concentration for linear DNA for the same extract is 2...
Here, we show that cell lysate prepared from E. coli BL21 Rosetta2 with a genomic knockout for either recB or recBCD operon supports high protein expression from linear DNA templates. This protocol elaborates a step-by-step lysate calibration procedure specific for linear DNA templates (Figure 2), which is a critical step that leads to the high expression from σ70 promoters in linear DNA, reaching near-plasmid levels for equimolar DNA concentrations. This prot...
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ACB and JLF acknowledge funding support by the ANR SINAPUV grant (ANR-17-CE07-0046). JLF and JB acknowledge funding support by the ANR SynBioDiag grant (ANR-18-CE33-0015). MSA and JLF acknowledge funding support by the ANR iCFree grant (ANR-20-BiopNSE). JB acknowledges support from the ERC starting "COMPUCELL" (grant number 657579). The Centre de Biochimie Structurale acknowledges support from the French Infrastructure for Integrated Structural Biology (FRISBI) (ANR-10-INSB-05-01). MK acknowledges funding support from INRAe's MICA department, Université Paris-Saclay, Ile-de-France (IdF) region's DIM-RFSI, and ANR DREAMY (ANR-21-CE48-003). This work was supported by funding through the European Research Council Consolidator Award (865973 to CLB).
Name | Company | Catalog Number | Comments |
2-YT Broth | Invitrogen | 22712020 | |
1.5 mL Safe-Lock tubes | Eppendorf | 30120086 | 1.5 mL microtube |
384-well square-bottom microplate | Thermo Scientific Nunc | 142761 | |
3PGA (D-(-)-3-Phosphoglyceric acid disodium) | Sigma-Aldrich | P8877 | |
adhesive plate seal | Thermo Scientific Nunc | 232701 | |
Agar | Invitrogen | 30391023 | |
ATP (Adenosine 5'-triphosphate disodium salt hydrate) | Sigma-Aldrich | A8937 | |
BL21 Rosetta2 | Merck Millipore | 71402 | |
BL21 Rosetta2 ΔrecB | Addgene | 176582 | |
BL21 Rosetta2 ΔrecBCD | Addgene | 176583 | |
cAMP (Adenosine 3' 5'-cyclic monophosphate) | Sigma-Aldrich | A9501 | |
Chloramphenicol | Sigma-Aldrich | C0378 | |
CoA (Coenzyme A hydrate) | Sigma-Aldrich | C4282 | |
Corning 15 mL PP Centrifuge Tubes, Rack Packed with CentriStar Cap, Sterile | Corning | 430790 | 15 mL tube |
Corning 50 mL PP Centrifuge Tubes, Conical Bottom with CentriStar Cap, Sterile | Corning | 430828 | 50 mL tube |
CTP (Cytidine 5'-triphosphate, disodium salt hydrate) | Alfa Aesar | J62238 | |
DpnI | NEB | R0176S | |
DTT (DL-Dithiothreitol) | Sigma-Aldrich | D0632 | |
Folinic acid (solid folinic acid calcium salt) | Sigma-Aldrich | F7878 | |
GTP (Guanosine 5ʹ-Triphosphate, Disodium Salt) | Sigma-Aldrich | 371701 | |
HEPES | Sigma-Aldrich | H3375 | |
K phosphate dibasic (K2HPO4) | Carl Roth | 231-834-5 | |
K phosphate monobasic (H2KO4P) | Sigma-Aldrich | P5655 | |
K-glutamate | Alfa Aesar | A17232 | |
Mg-glutamate | Sigma-Aldrich | 49605 | |
Millex-GP Syringe Filter Unit, 0.22 µm, polyethersulfone | Merck Millipore | SLGP033RB | membrane filter 0.22 µm |
Monarch PCR & DNA Cleanup Kit | NEB | T1030S | PCR & DNA cleanup Kit |
Monarch Plasmid Miniprep Kit | NEB | T1010S | Plasmid Miniprep Kit |
NAD (B-nicotinamide adenine dinucleotide hydrate) | Sigma-Aldrich | N6522 | |
NIST-traceable FITC standard | Invitrogen | F36915 | NIST-FITC |
NucleoBond Xtra Maxi kit | Macherey-Nagel | 740414.1 | Plasmid Maxiprep Kit |
PEG 8000 | Sigma-Aldrich | 89510 | |
plate reader | Biotek | Synergy HTX | |
Purified Recombinant EGFP Protein | Chromotek | egfp-250 | recombinant eGFP |
Q5 High-Fidelity 2X Master Mix | NEB | M0492S | DNA polymerase |
Q5 High-Fidelity 2X Master Mix | New England Biolabs | M0492L | DNA polymerase |
Qubit dsDNA BR Assay Kit | Thermo | Q32850 | fluorometric assay with DNA-binding dye |
RTS Amino Acid Sampler | biotechrabbit | BR1401801 | |
Spermidine | Sigma-Aldrich | 85558 | |
Sterile water | Purified water from the Millipore RiOs 8 system, sterilized by autoclaving. | ||
Tris base | Life Science products Cytiva | 17-1321-01 | |
tRNA | Roche | 10109550001 | |
Ultrasonic processor Vibra cell VC-505 | SONICS | VC505 | sonicator |
UTP Na3 (Uridine 5'- triphosphate, trisodium salt hydrate) | Acros Organics | 226310010 | |
Water, nuclease-free | Thermo | R0581 | nuclease-free water |
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