The authors acknowledge the Office of the Secretary of Defense&#8217;s Applied Research for the Advancement of S&#38;T Priorities program for supporting the collaboration that led to this collection.

<ol>
	<li>Silverman, A. D., Karim, A. S., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+gene+expression:+an+expanded+repertoire+of+applications.">Cell-free gene expression: an expanded repertoire of applications.</a> Nature Reviews. Genetics. 21 (3), 151-170 (2020).</li><li>Grasemann, L., Lavickova, B., Elizondo-Cant&#250;, M. C., Maerkl, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OnePot+PURE+cell-free+system.">OnePot PURE cell-free system.</a> Journal of Visualized Experiments. (172), e62625 (2021).</li><li>Smith, P. E. J., Slouka, T., Oza, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cells+to+cell-free+protein+synthesis+within+24+hours+using+cell-free+autoinduction+workflow.">From cells to cell-free protein synthesis within 24 hours using cell-free autoinduction workflow.</a> Journal of Visualized Experiments. (173), e62866 (2021).</li><li>Cooper, R. M., Tonooka, T., Didovyk, A., Hasty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+affordable,+and+uncomplicated+production+of+bacterial+cell-free+lysate.">Rapid, affordable, and uncomplicated production of bacterial cell-free lysate.</a> Journal of Visualized Experiments. (176), e62753 (2021).</li><li>Dopp, J. L., Reuel, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+enzymatic+methods+for+amplification+of+minimal,+linear+templates+for+protein+prototyping+using+cell-Free+Systems.">Rapid, enzymatic methods for amplification of minimal, linear templates for protein prototyping using cell-Free Systems.</a> Journal of Visualized Experiments. (172), e62728 (2021).</li><li>McManus, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+characterization+of+genetic+parts+with+cell-free+systems.">Rapid characterization of genetic parts with cell-free systems.</a> Journal of Visualized Experiments. (174), e62816 (2021).</li><li>Toh, M., Chengan, K., Hanson, T., Freemont, P. S., Moore, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-yield+streptomyces+transcription-translation+toolkit+for+synthetic+biology+and+natural+product+applications.">A high-yield streptomyces transcription-translation toolkit for synthetic biology and natural product applications.</a> Journal of Visualized Experiments. (175), e63012 (2021).</li><li>Dinglasan, J. L. N., Reeves, D. T., Hettich, R. L., Doktycz, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+chromatography+coupled+to+refractive+index+or+mass+spectrometric+detection+for+metabolite+profiling+in+lysate-based+Cell-free+systems.">Liquid chromatography coupled to refractive index or mass spectrometric detection for metabolite profiling in lysate-based Cell-free systems.</a> Journal of Visualized Experiments. (175), e62852 (2021).</li><li>Drachuk, I., Harbaugh, S., Kelley-Loughnane, N., Ch&#225;vez, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+multifunctional+silk-based+microcapsules+loaded+with+DNA+plasmids+encoding+RNA+aptamers+and+riboswitches.">Preparation of multifunctional silk-based microcapsules loaded with DNA plasmids encoding RNA aptamers and riboswitches.</a> Journal of Visualized Experiments. (176), e62854 (2021).</li><li>Jaenes, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+to+implementation+study+for+development+and+patient+validation+of+paper-based+toehold+switch+diagnostics.">Design to implementation study for development and patient validation of paper-based toehold switch diagnostics.</a> Journal of Visualized Experiments. (184), e63223 (2022).</li></ol>

The authors have nothing to disclose.

Cell-free expression (CFE) systems are a suite of tools that reconstitute the key cellular processes of transcription, translation, and metabolism outside the confines of the cell1. Such methods have grown rapidly in popularity by offering simplified experimentation via the removal of cell membranes and reduced complexity via the removal of genomic DNA and the corresponding complexity of interactions between a genetic construct and the host. CFE systems are now used for fundamental research, sensing, biomanufacturing, and education. This methods collection presents nine examples of new ways to produce and use cell-free systems.

Three of the methods focus on making the production of cell-free systems more accessible. Grasemann et al. describe an easier way to make the PURE system, a CFE system that reconstitutes&#160;all of the components required for transcription and translation, rather than using cellular lysates2. Instead of expressing and purifying each of the 36 protein components, the authors pool the expression strains, offering production of a functional PURE system within a week. Smith et al. offer a simplified process for the production of lysate-based CFE systems using a streamlined culturing approach that cuts the total workflow to 24 h, accomplishable within normal working hours3. Cooper et al. describe a method using a modified E. coli strain that enables&#160;cell lysis by freeze-thaw, eliminating the need for the specialized equipment required by other methods4. All of these methods contribute to a trend of efforts to make the production of cell-free systems attainable by labs with less infrastructure and expertise in the complicated traditional protocols. With the availability of competing commercial kits, more and more labs can explore the advantages of CFE systems with little risk. With methods such as those presented here, these labs can then choose to make their own CFE systems that are tailored to their needs without a huge investment in equipment or time.

Two methods focus on the advantages of the rapid and high-throughput experimentation possible with CFE systems. Dopp et al. present a method to amplify linear DNA templates for rapid screening of genetic constructs5. Using their approach, genetic designs can be ordered from DNA synthesis companies&#160;and then amplified and screened within 24 h. McManus et al. describe a similar approach whereby a reporter gene is amplified with varying primers encoding for different regulatory components6. The low cost of oligos paired with CFE systems enables rapid, cost-effective screening of components such as promoters. Both methods highlight the potential of CFE systems for rapid experimentation, reducing timelines for designing and testing a construct from weeks using traditional cloning to days or even hours.

The next three methods provide tools for specialized uses of CFE systems. Toh et al. offer a method for creating CFE systems from Streptomyces venezuelae7. A majority of&#160;CFE protocols are based on E. coli, including all other methods in this collection. This new method for S. venezuelae brings the advantages of CFE systems to an organism of broad interest for natural product production. Dinglasan et al. provide a way to measure metabolite concentrations in cell-free reactions8. Such measurements are critical toward applications in cell-free metabolic engineering, where the intact metabolism of CFE systems is used to produce non-protein products of interest. Third, Drachuk et al. present microencapsulation of DNA for addition to CFE reactions9. This approach is representative of a set of techniques that enables the advantages of compartmentalization to be rationally re-introduced into CFE systems after the removal of natural membranes during the manufacture of CFE systems. Each of these methods diversifies the range of applications that CFE systems can be applied to.

Finally, Jaenes et al. describe methods for producing a real-world diagnostic for Zika virus based on CFE systems10. The tests and instrumentation are low cost compared to competing technologies such as PCR, and the authors have successfully tested their diagnostics using patient samples in multiple countries. This work is a strong example of point-of-need sensing applications, which is perhaps the area where CFE systems is poised to make the most immediate real-world impact.

As a collection, these methods capture a representative swath of the main areas of active research in CFE systems in recent years. The methods that improve the production of CFE systems and enable high throughput experimentation are well-represented, and examples of the numerous more specific uses of CFE systems are present. Other areas of interest include the production of complex proteins for medical applications, incorporation of non-canonical amino acids, optimization for long-term storage, and development of artificial cells. We anticipate the adoption of CFE systems to continue to accelerate given the reduced barrier to entry, potential for rapid experimentation, and wider range of capabilities, all enabled by methods such as those presented in this collection. The remaining challenges depend on the application space. For biomanufacturing, reduction of costs at scale by moving to cheaper substrates and improved recycling will be critical. For sensing, significant work building on the example of Jaenes et al. is needed to demonstrate robust performance in various point-of-need applications. Broadly, the field needs to continue to refine core techniques while expanding the repertoire of what can be done with CFE systems.

cell-free expression systems: methods of production and use

ARTICLES DISCUSSED:
Grasemann, L., Lavickova, B., Elizondo-Cant&#250;, M. C., Maerkl, S. J. OnePot PURE cell-free system. Journal of Visualized Experiments. (172), doi:10.3791/62625 (2021).
Smith, P. E. J., Slouka, T., Dabbas, M., Oza, J. P. From cells to cell-free protein synthesis within 24 hours using cell-free autoinduction workflow. Journal of Visualized Experiments. (173), doi:10.3791/62866 (2021).
Cooper, R. M., Tonooka, T., Didovyk, A., Hasty, J. Rapid, affordable, and uncomplicated production of bacterial cell-free lysate. Journal of Visualized Experiments. (176), doi:10.3791/62753 (2021).
Dopp, J. L., Reuel, N. F. Rapid, enzymatic methods for amplification of minimal, linear templates for protein prototyping using cell-free systems. Journal of Visualized Experiments. (172), doi:10.3791/62728 (2021).
McManus, J. B. et al. Rapid characterization of genetic parts with cell-free systems. Journal of Visualized Experiments. (174), doi:10.3791/62816 (2021).
Toh, M., Chengan, K., Hanson, T., Freemont, P. S., Moore, S. J. A high-yield streptomyces transcription-translation toolkit for synthetic biology and natural product applications. Journal of Visualized Experiments. (175), doi:10.3791/63012 (2021).
Dinglasan, J. L. N., Reeves, D. T., Hettich, R. L., Doktycz, M. J. Liquid chromatography coupled to refractive index or mass spectrometric detection for metabolite profiling in lysate-based cell-free systems. Journal of Visualized Experiments. (175), doi:10.3791/62852 (2021).
Drachuk, I., Harbaugh, S., Kelley-Loughnane, N., Ch&#225;vez, J. L. Preparation of multifunctional silk-based microcapsules loaded with DNA plasmids encoding RNA aptamers and riboswitches. Journal of Visualized Experiments. (176), doi:10.3791/62854 (2021).
Jaenes, K. et al. Design to implementation study for development and patient validation of paper-based toehold switch diagnostics. Journal of Visualized Experiments. (184), doi:10.3791/63223 (2022).

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