Name: cell-free protein synthesis from exonuclease-deficient cellular extracts utilizing linear dna templates
Uploaded: 2022-08-09T14:00:00.000+00:00
Duration: 8 min 11 s
Description: Watch this Scientific Journal Video about Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates at JoVE.com

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 &#34;COMPUCELL&#34; (grant number 657579). The Centre de Biochimie Structurale acknowledges support from&#160;the French Infrastructure for Integrated Structural Biology (FRISBI) (ANR-10-INSB-05-01). MK acknowledges funding support from INRAe&#39;s MICA department, Universit&#233; Paris-Saclay, Ile-de-France (IdF) region&#39;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).

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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 &#963;70 promoters in linear DNA, reaching near-plasmid levels for equimolar DNA concentrations. This protocol highlights the importance of buffer calibration according to the type of DNA (linear or plasmid) to be used in the final application.
The protocol can help avoid plasmid cloning, which provides significant gains in time and cost. Linear DNA fragments can be synthesized by commercial providers and/or amplified by PCR in the laboratory. The same process using plasmid DNA would take at least 1 week longer, taking into account cloning, sequence verification, and DNA preparation steps10. It is recommended to prepare large volumes of the cell extract and linear DNA templates to minimize batch-to-batch variability between experiments14,15. 
This method has been shown to be robust and used across laboratories10. Results from these extracts have been consistent across biological replicates, different extract batches, as well as across different strain backgrounds10. The robustness of the protocol has also been tested across different experimental parameters: cell lysis methods, temperatures, DNA templates, and different DNA concentrations. The method has already been used for rapid screening and characterization of genetic constructs for two relevant applications: toehold switch library characterization and enzyme activity screening10. 
Although high expression levels were obtained in this study from linear DNA in &#916;recB/&#916;recBCD extracts from the highly productive parental strain E. coli BL21 Rosetta2, the methodology would require genome engineering for a new strain of interest. In contrast, supplementation with protective agents for linear DNA, like GamS5 or Chi DNA6, is easier to apply for a new strain, but costlier and more time-consuming.
The protocol provided here will facilitate the use of linear DNA templates in cell-free systems and accelerate the design-build-test cycles for the synthetic biology community. Easier cell-free expression from linear DNA templates in &#916;recB/&#916;recBCD extracts can be deployed for the production of small molecules of interest, biosimilars, and other on-demand point-of-care applications.

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&#160;cloning and&#160;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 &#955;-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 &#916;recBCD 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 &#963;70 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 &#916;recBCD cell extracts and specific calibration for linear DNA as template.

<table><tbody><tr><td>2-YT Broth</td><td>Invitrogen</td><td>22712020</td><td></td></tr><tr><td>1.5 mL Safe-Lock tubes</td><td>Eppendorf</td><td>30120086</td><td>1.5 mL microtube</td></tr><tr><td>384-well square-bottom microplate</td><td>Thermo Scientific Nunc</td><td>142761</td><td></td></tr><tr><td>3PGA (D-(-)-3-Phosphoglyceric acid disodium)</td><td>Sigma-Aldrich</td><td>P8877</td><td></td></tr><tr><td>adhesive plate seal</td><td>Thermo Scientific Nunc</td><td>232701</td><td></td></tr><tr><td>Agar</td><td>Invitrogen</td><td>30391023</td><td></td></tr><tr><td>ATP (Adenosine 5&#039;-triphosphate disodium salt hydrate)</td><td>Sigma-Aldrich</td><td>A8937</td><td></td></tr><tr><td>BL21 Rosetta2</td><td>Merck Millipore</td><td>71402</td><td></td></tr><tr><td>BL21 Rosetta2 &amp;Delta;recB</td><td>Addgene</td><td>176582</td><td></td></tr><tr><td>BL21 Rosetta2 &amp;Delta;recBCD</td><td>Addgene</td><td>176583</td><td></td></tr><tr><td>cAMP (Adenosine 3&#039; 5&#039;-cyclic monophosphate)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A9501</td><td></td></tr><tr><td>Chloramphenicol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C0378</td><td></td></tr><tr><td>CoA (Coenzyme A hydrate)</td><td>Sigma-Aldrich</td><td>C4282</td><td></td></tr><tr><td>Corning 15 mL PP Centrifuge Tubes, Rack Packed with CentriStar Cap, Sterile</td><td>Corning</td><td>430790</td><td>15 mL tube</td></tr><tr><td>Corning 50 mL PP Centrifuge Tubes, Conical Bottom with CentriStar Cap, Sterile</td><td>Corning</td><td>430828</td><td>50 mL tube</td></tr><tr><td>CTP (Cytidine 5&#039;-triphosphate, disodium salt hydrate)</td><td>Alfa Aesar</td><td>&amp;nbsp;J62238</td><td></td></tr><tr><td>DpnI</td><td>NEB</td><td>R0176S</td><td></td></tr><tr><td>DTT (DL-Dithiothreitol)</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>Folinic acid (solid folinic acid calcium salt)</td><td>Sigma-Aldrich</td><td>F7878</td><td></td></tr><tr><td>GTP (Guanosine 5?-Triphosphate,&amp;nbsp; Disodium Salt)</td><td>Sigma-Aldrich</td><td>371701</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>&amp;nbsp;H3375</td><td></td></tr><tr><td>K phosphate dibasic (K2HPO4)</td><td>Carl Roth</td><td>231-834-5</td><td></td></tr><tr><td>K phosphate monobasic (H2KO4P)</td><td>Sigma-Aldrich</td><td>P5655</td><td></td></tr><tr><td>K-glutamate</td><td>Alfa Aesar</td><td>A17232</td><td></td></tr><tr><td>Mg-glutamate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>49605</td><td></td></tr><tr><td>Millex-GP Syringe Filter Unit, 0.22 &amp;micro;m, polyethersulfone</td><td>Merck Millipore</td><td>SLGP033RB</td><td>membrane filter 0.22 &amp;micro;m</td></tr><tr><td>Monarch PCR &amp;amp; DNA Cleanup Kit</td><td>NEB</td><td>T1030S</td><td>PCR &amp;amp; DNA cleanup Kit</td></tr><tr><td>Monarch Plasmid Miniprep Kit</td><td>NEB</td><td>T1010S</td><td>Plasmid Miniprep Kit</td></tr><tr><td>NAD (B-nicotinamide adenine dinucleotide hydrate)&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>N6522</td><td></td></tr><tr><td>NIST-traceable FITC standard</td><td>Invitrogen</td><td>F36915</td><td>NIST-FITC</td></tr><tr><td>NucleoBond Xtra Maxi kit</td><td>Macherey-Nagel</td><td>740414.1</td><td>Plasmid Maxiprep Kit</td></tr><tr><td>PEG 8000</td><td>Sigma-Aldrich</td><td>89510</td><td></td></tr><tr><td>plate reader</td><td>Biotek&amp;nbsp;</td><td>Synergy HTX</td><td></td></tr><tr><td>Purified Recombinant EGFP Protein</td><td>Chromotek</td><td>egfp-250</td><td>recombinant eGFP</td></tr><tr><td>Q5 High-Fidelity 2X Master Mix</td><td>NEB</td><td>M0492S</td><td>DNA polymerase</td></tr><tr><td>Q5 High-Fidelity 2X Master Mix</td><td>New England Biolabs</td><td>M0492L</td><td>DNA polymerase</td></tr><tr><td>Qubit dsDNA BR Assay Kit</td><td>Thermo</td><td>Q32850</td><td>fluorometric assay with DNA-binding dye</td></tr><tr><td>RTS Amino Acid Sampler</td><td>biotechrabbit</td><td>BR1401801</td><td></td></tr><tr><td>Spermidine</td><td>Sigma-Aldrich</td><td>85558</td><td></td></tr><tr><td>Sterile water</td><td></td><td></td><td>Purified water from the Millipore RiOs 8 system, sterilized by autoclaving.</td></tr><tr><td>Tris base</td><td>Life Science products Cytiva</td><td>17-1321-01&amp;nbsp;</td><td></td></tr><tr><td>tRNA</td><td>Roche</td><td>10109550001</td><td></td></tr><tr><td>Ultrasonic processor Vibra cell VC-505</td><td>SONICS</td><td>VC505</td><td>sonicator</td></tr><tr><td>UTP Na3 (Uridine 5&#039;- triphosphate, trisodium salt hydrate)</td><td>Acros Organics&amp;nbsp;</td><td>226310010</td><td></td></tr><tr><td>Water, nuclease-free</td><td>Thermo</td><td>R0581</td><td>nuclease-free water</td></tr></tbody></table>

cell-free protein synthesis from exonuclease-deficient cellular extracts utilizing 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 &#916;recBCD 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.

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 &#916;recB and &#916;recBCD extracts at 8 mM (Figure 1B). However, the optimal K-glutamate concentration for plasmid DNA is 140&#160;mM, whereas the optimal K-glutamate concentration for linear DNA for the same extract is 20 mM (Figure 1C). In a comparative analysis of GFP expression in extracts from WT and &#916;recBCD cells, it was seen that the buffer composition of extracts must be specifically calibrated for optimal expression from linear and plasmid DNA used in exonuclease V deleted extracts.
After calibration steps, levels of GFP expressions were compared from linear and plasmid DNA (5 nM) in the WT and &#916;recBCD extract (Figure 1D). GFP expression from linear DNA reached 102% and 138% of the expression from plasmid DNA in extracts from &#916;recB and &#916;recBCD strains, respectively. Together, these results show that expression from linear DNA, using a native E. coli&#160;&#963;70 promoter, can reach the same level as that from plasmid expression when the cell lysate is prepared from such mutants and the cell-free buffer is specifically calibrated for linear DNA.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64236/64236fig1v2.jpg" /> 
Figure 1: Differential buffer optimization for linear and plasmid DNA in &#916;recB/&#916;recBCD extracts. (A) A 1361 bp linear DNA amplicon was amplified from plasmid p70a-deGFP and used as a template for gene expression for buffer calibration. (B) Mg-glutamate buffer calibration with 1 nM of each DNA type using different concentrations from 0 to 20 mM (with K-glutamate fixed at 80 mM). Square boxes indicate concentration points chosen. P = plasmid DNA, L = linear DNA. (C) After selecting the optimal Mg-glutamate concentration for each lysate, K-glutamate was titrated from 20 to 240 mM. Square boxes indicate concentration points chosen. P = plasmid DNA, L = linear DNA. Data shown in the heat-maps (B) and (C) are from a single experiment. (D) After buffer calibration, cell-free reactions were performed with 5 nM of each DNA type. Extract BL21 Rosetta2 does not support linear DNA expression, whereas differentially optimized extracts from &#916;recB and &#916;recBCD strains exhibit linear DNA (green) expression similar to plasmid DNA (blue) levels. Endpoint data shown are mean &#177; SD for three replicates done on the same day and collected after 8 h of incubation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64236/64236fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64236/64236fig2v2.jpg" /> 
Figure 2: Visual representation of the protocol. Workflow for cell lysate preparation and lysate optimization for linear DNA as a template in CFPS synthesis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64236/64236fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: List of components for preparation of liquid and solid 2xYT+P media. The quantity (in grams) of each component is specified for 1 L of media. Scale up proportionally as needed. Media must be autoclaved before use. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64236/JoVE_LinDNA_Table_1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: List of components for preparation of buffer S30A+DTT. The quantity (in grams) of each component is specified for 1 L of buffer S30A. Scale up proportionally as needed. S30A buffer must be autoclaved and then chilled (4 &#176;C) before use. The DTT buffer must be prepared and filtered separately as indicated (in 15 mL tube) and stored in -20 &#176;C. Right before using buffer S30A, add 2 mL of DTT (from 1 M stock) to 1 L of S30A buffer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64236/JoVE_LinDNA_Table_2.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: List of components for energy solution preparation. The quantity (in grams) or the volume of each component is specified (in column G) for the preparation of a specified volume (in column H) of stock solutions for the indicated concentration (in column F). The pH of each component must be adjusted if needed, as it is indicated (in column I and J). 14x energy solution stock is prepared by mixing the listed volume of each component (in column K) in the order listed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64236/JoVE_LinDNA_Table_3.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4: List of components for preparation of amino acid stock solution. For preparation of the 4x amino acid stock solution, all of the amino acids must be mixed in the order listed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64236/JoVE_LinDNA_Table_4.xlsx" target="_blank">Please click here to download this Table.</a>
Table 5. List of components for preparation of lysate calibration solutions. The quantity (in grams) of Mg-glutamate and K-glutamate stock solutions are specified to prepare 50 mL of the indicated concentration of each buffer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64236/JoVE_LinDNA_Table_5.xlsx" target="_blank">Please click here to download this Table.</a>
Table 6: List of bacterial strains used in this protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64236/JoVE_LinDNA_Table_6.xlsx" target="_blank">Please click here to download this Table.</a>
Table 7: List of primers used in this protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64236/JoVE_LinDNA_Table_7.xlsx" target="_blank">Please click here to download this Table.</a>
Table 8: Plasmid used in this protocol for lysate calibration and as a template for linear DNA amplification by PCR. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64236/JoVE_LinDNA_Table_8.xlsx" target="_blank">Please click here to download this Table.</a>
Table 9: FITC and eGFP conversion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64236/JoVE_LinDNA_Table_9.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64236/JoVE_SupFile_1_TXTL_Linear_Plasmid_calibration.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Figure 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64236/JoVE_SupFig_1.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates at JoVE.com

Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates

Presented here is a protocol for the preparation and buffer calibration of cell extracts from exonuclease V knockout strains of Escherichia coli BL21 Rosetta2 (&#916;recBCD and &#916;recB). 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 ...

cell-free-protein-synthesis-from-exonuclease-deficient-cellular

Nanyang Technological University

Research

JoVE Journal

Bioengineering

3.6K Views.  Université Paris-Saclay. Het protocol is zeer belangrijk omdat het snel, eenvoudig en direct gebruik van lineaire DNA-sjablonen in celvrije eiwitsynthesesystemen mogelijk maakt, zelfs van inheemse E.coli-parameters. De belangrijkste voordelen zijn de tijdshoeveelheid die wordt bespaard door het vermijden van klonen, chemische modificaties van lineaire DNA-uiteinden en beschermende supplementen zoals gamma- of chi-DNA. Aangezien zelfrapportagesynthese-extracten worden ontwikkeld voor verschillende organismen van belan...

Video: Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up cellular mimics that better represent the complexity of cellular life. To date there are a number of paths that could be taken to build compartmentalized cellular mimics, including the exploitation of water-in-oil emulsions, microfluidic devices, and vesicles. Each of the available options has specific advantages and disadvantages. For example, water-in-oil emulsions give high encapsulation efficiency but do not mimic well the permeability barrier of living cells. The primary advantage of the methods described herein is that they are all easy and cheap to implement. Transcription-translation machinery is encapsulated inside of phospholipid vesicles through a process that exploits common instrumentation, such as a centrifugal evaporator and an extruder. Reactions are monitored by fluorescence spectroscopy. The protocols can be adapted for recombinant protein expression, the construction of cellular mimics, the exploration of the minimum requirements for cellular life, or the assembly of genetic circuitry.

Herein we describe simple methods for the preparation of vesicles, the encapsulation of transcription and translation machinery, and the monitoring of protein production. The resulting cell-free systems can be used as a starting point from which to build increasingly complex cellular mimics.

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

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 &#181;g/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 &#181;g of protein produced or $0.019 per &#181;L 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.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

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 ...

Chemistry

CFPS Protocols for Micro-Compartmentalized Synthetic Cells

Cell-Free Protein Synthesis System for Building Synthetic Cells

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.

Author Spotlight: Optimizing CFPS Systems for Synthetic Cell Construction

This protocol describes the Cell-Free Protein Synthesis (CFPS) system used in constructing synthetic cells. It outlines key stages with representative results in different micro-compartments. The protocol aims to establish best practices for diverse laboratories in the synthetic cell community, advancing progress in synthetic cell development.

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for ...

Biochemistry

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

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 &#956;L batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of &#62; 260 &#956;g/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1&#8722;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 Expression Using the Rapidly Growing Bacterium Vibrio natriegens

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 ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 106x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 &#181;g of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.

The study describes a protocol for creating large (&#181;g-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins ...

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the ...

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

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.

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 ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates

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