The authors greatly acknowledge the support of the Biotechnology and Biological Sciences Research Council awards BB/V017551/1 (S.K., T.P.H.) and BB/W01095X/1 (A.L., T.P.H.), and the Engineering and Physical Sciences Research Council - Defence Science and Technology Laboratories award EP/N026683/1 (C.J.W., A.M.B., T.P.H.). Data supporting this publication are openly available at: 10.25405/data.ncl.22232452. For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) license to any Author Accepted Manuscript version arising.

1. Cell lysate buffer and media preparation
<ol>
	<li>Preparation of 2x YT+P agar and medium
	<ol>
		<li>Prepare 2x YT+P agar by measuring out 16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, 40 mL/L 1 M K2HPO4, 22 mL/L 1 M KH2PO4, and 15 g/L agar. For the 2x YT+P broth, follow the previous composition but omit the agar.</li>
		<li>Sterilize by autoclaving the 2x YT+P.</li>
	</ol>
	</li>
	<li>Preparation of the S30A buffer
	<ol>
		<li>Prepare the S30A buffer with 5.88 g/L Mg-glutamate, 12.195 g/L K-glutamate, and 25 mL/L 2 M Tris, adjusted to pH 7.7 with acetic acid.</li>
		<li>Store the S30A buffer at 4 &#176;C for up to 1 week.</li>
	</ol>
	</li>
	<li>Preparation of the S30B buffer
	<ol>
		<li>Prepare the S30B buffer with 5.88 g/L Mg-glutamate, and 12.195 g/L K-glutamate, adjusted to pH 8.2 with 2 M Tris.</li>
		<li>Store the S30B buffer at 4 &#176;C for up to 1 week.</li>
	</ol>
	</li>
</ol>
2. Cell lysate preparation (4 day protocol)
<ol>
	<li>Day 1
	<ol>
		<li>Pour the 2x YT+P into agar plates supplemented with 100 &#181;g/mL ampicillin.</li>
		<li>Streak E. coli from &#8722;80 &#176;C glycerol stock, and incubate overnight at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Inoculate a single colony from the 2x YT+P plate into 400 mL of 2x YT+P broth (supplemented with ampicillin) in a 1 L baffled flask.</li>
		<li>Incubate for 24 h at 37 &#176;C with 220 rpm shaking.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Measure and record the OD600 of the 24 h cultures; growth is sufficient at an OD600 of 3-4. 
		NOTE: Take a 1 mL aliquot of bacterial culture, and perform a serial dilution with medium (2x YT+P broth) prior to measuring the OD600 nm. Allow for the dilution effect when calculating the OD600.</li>
		<li>Transfer the culture evenly between pre-chilled 500 mL centrifuge bottles.</li>
		<li>Centrifuge at 4,500 x g for 15 min at 4 &#176;C, and then discard the supernatant.</li>
		<li>While centrifuging, complete the S30A buffer preparation by adding 2 mL/L of 1 M DTT to the previously prepared S30A buffer, mixing, and maintaining the buffer on ice.</li>
		<li>Resuspend the cell pellets in 200 mL of S30A buffer.</li>
		<li>Vortex the bottles vigorously until the whole pellet is completely solubilized, keeping the cells on ice as much as possible.</li>
		<li>Centrifuge at 4,500 x g for 15 min at 4 &#176;C, and then discard the supernatant.</li>
		<li>Repeat steps 2.3.5-2.3.7 (inclusive).</li>
		<li>Add 40 mL of S30A buffer to each centrifuge bottle, resuspend the pellets, and transfer to pre-weighed 50 mL centrifuge tubes.</li>
		<li>Centrifuge at 4,000 x g for 15 min at 4 &#176;C. Discard the supernatant by decanting, and remove the residual supernatant by pipette, keeping it on ice as much as possible.</li>
		<li>Re-weigh the centrifuge tubes, record the new mass, and calculate the mass of the pellets.</li>
		<li>Flash-freeze pellets in liquid nitrogen, and store them at &#8722;80 &#176;C.</li>
	</ol>
	</li>
	<li>Day 4
	<ol>
		<li>Thaw the pellets on ice.</li>
		<li>Per 1 g of pellet, add the following: 1.2 mL of S30A buffer (DTT added before use), 275 &#181;L of protease inhibitor (8 mM stock), and 25 &#181;L of lysozyme (10 mg/mL stock).</li>
		<li>Vortex vigorously until the pellets are completely solubilized with no remaining clumps, keeping them on ice as much as possible.</li>
		<li>In 50 mL centrifuge tubes, sonicate the cell suspensions at 120 W, 20 kHz, 30% amplitude, and 20 s/40 s on/off pulses for 5 min of sonication.</li>
		<li>Centrifuge at 4,000 x g for 1 h at 4 &#176;C to pellet the cell debris.</li>
		<li>Transfer the supernatant to fresh 50 mL centrifuge tubes.</li>
		<li>Incubate tubes at 37 &#176;C at 220 rpm for 80 min.</li>
		<li>Prepare dialysis materials by adding 1 mL/L of 1 M DTT to S30B buffer. Mix and add 900 mL to a 1 L sterile beaker. Add a magnetic stirrer, and keep it at 4 &#176;C.</li>
		<li>Following the runoff reaction, transfer the cell extract from step 2.4.7 into 2 mL microcentrifuge tubes, and centrifuge at 20,000 x g for 10 min at 4 &#176;C.</li>
		<li>Consolidate the pellet-free supernatant on ice in a 50 mL centrifuge tube.</li>
		<li>Determine the total volume of cell extract produced, and hydrate the necessary number of 10K MWCO dialysis cassettes by submerging them in S30B for 2 min.</li>
		<li>Load the cassettes via a syringe with up to 3 mL of extract. Dialyze up to three cassettes per 1 L beaker, stirring at 4 &#176;C for 3 h.</li>
		<li>After the dialysis is complete, which clarifies the extract, transfer the extract to 2 mL micro-centrifuge tubes. Centrifuge at 20,000 x g for 10 min at 4 &#176;C.</li>
		<li>Consolidate the clarified extract into a fresh centrifuge tube on ice, and vortex briefly to mix.</li>
		<li>Determine the protein concentration of the extract using a fluorometer.</li>
		<li>Dilute the extract to a concentration of 44.5 mg/mL using pre-chilled ddH2O.</li>
		<li>Aliquot into 200 &#181;L aliquots, flash-freeze in liquid nitrogen, and store at &#8722;80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Preparation of lyophilized hydrogels (Method A)
<ol>
	<li>Weigh 0.75 g of agarose, add ddH2O to a volume of 100 mL, and microwave in 30 s bursts at high temperatures to create molten agarose stock.</li>
	<li>Using a pipette, transfer 50 &#181;L of molten agarose to a 1.5 mL microcentrifuge tube, and allow the gel to polymerize for 20-30 min (polymerization occurs faster if the gels are transferred to a fridge).</li>
	<li>Flash-freeze the microcentrifuge tubes, containing the polymerized gels, in liquid nitrogen.</li>
	<li>Remove the lid of the centrifuge tubes, cover the centrifuge tube openings with wax film, and pierce the film several times with a needle or pipette tip.</li>
	<li>Transfer the microcentrifuge tubes containing the polymerized gels to &#8722;80 &#176;C storage for 1-2 h.</li>
	<li>Recover the centrifuge tubes from &#8722;80 &#176;C storage, and place in a freeze-drier, ensuring the tubes are completely dry.</li>
	<li>Engage the freeze-drier with the following settings: temperature: &#8722;20 &#176;C, pressure: 0.1 mbar.</li>
	<li>Leave the gel to freeze-dry overnight.</li>
	<li>Recover the gels from the freeze-drier, and place them into &#8722;80 &#176;C storage until required. 
	&#8203;NOTE: Gels can be stored freeze-dried for up to 1 year.</li>
</ol>
4. Preparation of the 14x energy solution stock
<ol>
	<li>Combine all the reaction components (Table 1) in a 15 mL centrifuge tube on ice to produce a 14x energy solution.</li>
	<li>Aliquot the 14x energy solution into 1.5 mL microcentrifuge tubes, and store at &#8722;80 &#176;C (smaller-volume aliquots can be used). 
	&#8203;NOTE: The 14x energy solution can be stored for several months at &#8722;80 &#176;C if repeated freeze-thawing of the tubes is avoided.</li>
</ol>
5. Preparation of the 4x amino acid stock
<ol>
	<li>Thaw, on ice, all 20 amino acid components of an RTS Amino Acid Sampler kit. Vortex the amino acids to ensure they are completely dissolved.</li>
	<li>In a 50 mL centrifuge tube, combine all 20 amino acids, and add 12 mL of sterile ddH2O. Vortex until the solution becomes clear, with only a slight haze of white coloration.</li>
	<li>Aliquot the 4x amino acids into 1.5 mL microcentrifuge tubes (500 &#181;L aliquots).</li>
	<li>Flash-freeze the 4x amino acid solution aliquots in liquid nitrogen, and move the aliquots to &#8722;80 &#176;C storage.</li>
</ol>
6. Cell-free buffer calibration
NOTE: The CFPS buffer used for the hydrogel reactions was calibrated for optimal DTT, Mg-glutamate, and K-glutamate concentrations following the protocol in Banks et al.34 modified from Sun et al.35. The calibration reaction compositions were selected using the design of experiments (DOE) method, with seven factor levels being selected for K-glutamate (200 mM, 400 mM, 600 mM, 1,000 mM, 1,200 mM, 1,400 mM), Mg-glutamate (0 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM), and DTT (0 mM, 5, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM) stocks. JMP Pro 15 was used to generate a custom DOE design (Table 2) and conduct the analysis to determine the optimal factor concentrations.
<ol>
	<li>Recover the cell-free extract from &#8722;80 &#176;C storage, and thaw on ice.</li>
	<li>Thaw the 4x amino acids, 14x energy solution, 40% PEG-8000, plasmid DNA, 3 M K-glutamate, 100 mM Mg-glutamate, and 100 mM DTT stocks on ice.</li>
	<li>Create a calibration master mix by combining 12.5 &#181;L of the 4x amino acids, 3.57 &#181;L of the 14x energy solution, 2.5 &#181;L of 40% PEG-8000, 3 &#181;g of plasmid DNA, and 10 &#181;L of cell-free extract (44.5 mg/mL), and make up to 35 &#181;L per reaction with ddH2O.</li>
	<li>Distribute the 35 &#181;L of master mix into the wells of a black 384-well microtiter plate.</li>
	<li>Following the DOE design, add the appropriate volume of Mg-glutamate, K-glutamate, and DTT to each reaction, along with ddH2O to make each reaction up to 50 &#181;L.</li>
	<li>Transfer the microtiter plate to a plate reader for fluorescence detection and analysis using the plate reader settings described (for mCherry): excitation: 587 nm, emission: 610 nm, wavelength bandwidth: 12 nm, optics: top, temperature: 37 &#176;C, with fluorescence readings being collected every 5 min for 4 h of total read time. Incubate the plates with shaking on a pulsed setting at 60 rpm.</li>
	<li>Upload the results into the JMP DOE design, and generate a prediction profile to determine the optimal concentration levels for K-glutamate, Mg-glutamate, and DTT based on desired response variables; in this case, the maximum fluorescence and reaction rate were the response variables.</li>
	<li>Combine the reagents as shown in Table 3 to create the 2X CFPS buffer</li>
	<li>Aliquot and store at &#8722;80 &#176;C</li>
</ol>
7. CFPS in rehydrated lyophilized hydrogels (Method A)
NOTE: The plasmids used in the CFPS reactions were created using components from the EcoFlex modular toolkit for E. coli36 and described in Whitfield et al.11&#160;The mCherry and eGFP were under the control of the constitutive JM23100 promoter. These components are available from Addgene.
<ol>
	<li>Recover the cell-free extract from &#8722;80 &#176;C storage, and thaw on ice for approximately 20 min.</li>
	<li>Collect the 2x CFPS buffer and plasmid DNA, and thaw on ice.</li>
	<li>Collect the freeze-dried hydrogels, and allow them to reach room temperature for 15 min by leaving the gels to stand on the bench.</li>
	<li>Transfer the gels to 1.5 mL microcentrifuge tubes, trimming the gels with a scalpel if necessary to make them fit in the wells.</li>
	<li>In a 1.5 mL microcentrifuge tube, combine 10 &#181;L of cell-free extract with 25 &#181;L of 2x CFPS buffer and 4 &#181;g of plasmid DNA; make up to a total volume of 50 &#181;L with ddH2O to create the CFPS solution.</li>
	<li>Pipette the CFPS solution onto the freeze-dried hydrogels.</li>
	<li>Allow the gels to soak in the CFPS system for 5-10 min at room temperature.</li>
	<li>Transfer the gels to a black 384-well microtiter plate using a spatula.</li>
	<li>Transfer the microtiter plate to a plate reader for fluorescence detection and analysis using the plate reader settings described in step 6.6. Representative results are available in previous studies31,33.</li>
</ol>
8. Cell-free protein synthesis in deployable hydrogels (Method B)
<ol>
	<li>Recover cell-free extract from &#8722;80 &#176;C storage, and thaw on ice for approximately 20 min.</li>
	<li>Collect the plasmid DNA, and thaw on ice.</li>
	<li>In a 1.5 mL microcentrifuge tube on ice, combine 10 &#181;L of cell-free extract with 3&#160;&#181;g of plasmid DNA and 25 &#181;L of 2x&#160;CFPS buffer,&#160;making&#160;up to a total volume of 37.5 &#181;L.</li>
	<li>Measure 3&#160;g of agarose, and add to 100 mL of ddH2O buffer to create 3% agarose.</li>
	<li>Microwave the 3% agarose in 30 s bursts at high power.</li>
	<li>Pipette 12.5&#160;&#181;L of molten agarose into 1.5 mL microcentrifuge tubes containing CFPS mix to a total volume of 50 &#181;L.</li>
	<li>Allow the molten agarose to cool, but not polymerize, leaving the agarose in a heat block set to 50 &#176;C.</li>
	<li>Combine the molten agarose with the CFPS solution; mix via pipetting and stirring with the tip, and ensure to move quickly to avoid gel polymerization before the CFPS solution can be mixed in.</li>
	<li>Allow the gels to cool at room temperature and polymerize for approximately 2 min.</li>
	<li>Transfer the polymerized agarose to 1.5 mL microcentrifuge tubes with a spatula, and flash-freeze in liquid nitrogen.</li>
	<li>Place the flash-frozen hydrogels into &#8722;80 &#176;C storage for 1 h.</li>
	<li>Recover the gels from storage; remove the microcentrifuge tube lids, cover the tubes with wax film, and pierce the film to allow the moisture to be dried off.</li>
	<li>Engage the freeze-drier with the following settings: temperature: &#8722;20 &#176;C, pressure: 0.1 mbar, freeze drying for 18 h (overnight).</li>
	<li>Store the freeze-dried CFPS devices in &#8722;80 &#176;C storage until use.</li>
	<li>The freeze-dried CFPS devices, with an approximate wet volume of 50 &#181;L, can be rehydrated with 50 &#181;L of ddH2O without excess liquid. Rehydration takes approximately 30 min.</li>
	<li>Transfer the gels to a black 384-well microtiter plate using a spatula.</li>
	<li>Transfer the microtiter plate to a plate reader for fluorescence detection and analysis using the plate reader settings described in step 6.6. Representative results are available in previous publications31,33.</li>
</ol>

Embedding CFPS Reactions in a Variety of Hydrogel Matrices

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A., 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=Agarose-based+biomaterials:+Opportunities+and+challenges+in+cartilage+tissue+engineering.">Agarose-based biomaterials: Opportunities and challenges in cartilage tissue engineering.</a> Polymers. 12 (5), 1150 (2020).</li><li>Buddingh, B. C., Van Hest, J. C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+cells:+Synthetic+compartments+with+life-like+functionality+and+adaptivity.">Artificial cells: Synthetic compartments with life-like functionality and adaptivity.</a> Accounts of Chemical Research. 50 (4), 769-777 (2017).</li><li>Kahn, J. S., 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=DNA+microgels+as+a+platform+for+cell-free+protein+expression+and+display.">DNA microgels as a platform for cell-free protein expression and display.</a> Biomacromolecules. 17 (6), 2019-2026 (2016).</li><li>Yang, D., 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=Enhanced+transcription+and+translation+in+clay+hydrogel+and+implications+for+early+life+evolution.">Enhanced transcription and translation in clay hydrogel and implications for early life evolution.</a> Scientific Reports. 3, 3165 (2013).</li><li>Zhou, X., Wu, H., Cui, M., Lai, S. N., Zheng, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-lived+protein+expression+in+hydrogel+particles:+Towards+artificial+cells.">Long-lived protein expression in hydrogel particles: Towards artificial cells.</a> Chemical Science. 9 (18), 4275-4279 (2018).</li><li>Whitfield, C. J., 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=Cell-free+genetic+devices+confer+autonomic+and+adaptive+properties+to+hydrogels.">Cell-free genetic devices confer autonomic and adaptive properties to hydrogels.</a> BioRxiv. , (2019).</li><li>Feng, L., Jianpu, T., Jinhui, G. D., Luo, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+DNA+hydrogel:+Design,+synthesis+and+applications.">Polymeric DNA hydrogel: Design, synthesis and applications.</a> Progress in Polymer Science. 98, 101163 (2019).</li><li>Howard, T., 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=Datasets+for+Whitfield+et+al.">Datasets for Whitfield et al.</a> 2020 Chemical Communications. , (2020).</li><li>Banks, A. M., 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=Key+reaction+components+affect+the+kinetics+and+performance+robustness+of+cell-free+protein+synthesis+reactions.">Key reaction components affect the kinetics and performance robustness of cell-free protein synthesis reactions.</a> Computational and Structural Biotechnology Journal. 20, 218-229 (2022).</li><li>Sun, Z. Z., 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=Protocols+for+implementing+an+Escherichia+coli-based+TX-TL+cell-free+expression+system+for+synthetic+biology.">Protocols for implementing an Escherichia coli-based TX-TL cell-free expression system for synthetic biology.</a> Journal of Visualized Experiments. (79), e50762 (2013).</li><li>Moore, S. J., 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=EcoFlex:+A+multifunctional+MoClo+kit+for+E.+coli+synthetic+biology.">EcoFlex: A multifunctional MoClo kit for E. coli synthetic biology.</a> ACS Synthetic Biology. 5 (10), 1059-1069 (2016).</li><li>Ben&#237;tez-Mateos, A. I., 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=Micro+compartmentalized+cell-free+protein+synthesis+in+hydrogel+&#181;-channels.">Micro compartmentalized cell-free protein synthesis in hydrogel &#181;-channels.</a> ACS Synthetic Biology. 9 (11), 2971-2978 (2020).</li></ol>

Outlined here are two protocols for the incorporation of E. coli cell lysate-based CFPS reactions into agarose hydrogels. These methods allow for simultaneous gene expression throughout the material. The protocol can be adapted for other CFPS systems and has been successfully conducted with commercially available CFPS kits in addition to the laboratory-prepared cell lysates detailed here. Importantly, the protocol allows for gene expression in the absence of an external liquid phase. This means that the system i...

Synthetic biology integrates diverse engineering disciplines to design and engineer biologically based parts, devices, and systems that can perform functions that are not found in nature. Most synthetic biology approaches are still bound to living cells. By contrast, cell-free synthetic biology systems facilitate unprecedented levels of control and freedom in design, enabling increased flexibility and a shortened time for engineering biological systems while eliminating many of the constraints of traditional cell-based gene expression methods1,2,3. CFPS is being used in an increasing number of applications across numerous disciplines, including constructing artificial cells, prototyping genetic circuits, developing biosensors, and producing metabolites4,5,6. CFPS has also been particularly useful for producing recombinant proteins that cannot be readily expressed in living cells, such as aggregation-prone proteins, transmembrane proteins, and toxic proteins6,7,8.
CFPS is typically performed in liquid reactions. This, however, may restrict their deployment in some situations, as any liquid cell-free device must be contained within a reaction vessel. The rationale for the development of the methods presented here was to provide robust protocols for embedding cell-free synthetic biology devices into hydrogels, not as a protein production platform per se but, instead, to allow the use of hydrogels as a physical chassis for the deployment of cell-free devices beyond the laboratory. The use of hydrogels as CFPS chassis has several advantages. Hydrogels are polymeric materials that, despite a high water content (sometimes in excess of 98%), possess solid properties9,10,11. They have uses as pastes, lubricants, and adhesives and are present in products as diverse as contact lenses, wound dressings, marine adhesive tapes, soil improvers, and baby diapers9,11,12,13,14. Hydrogels are also under active investigation as drug delivery vehicles9,15,16,17. Hydrogels may also be biocompatible, biodegradable, and possess some stimuli responses of their own9,18,19,20. Therefore, the goal here is to create a synergy between molecular biology-derived functionality and materials science. To this end, efforts have been made to integrate cell-free synthetic biology with a range of materials, including collagen, laponite, polyacrylamide, fibrin, PEG-peptide, and agarose11,21,22, as well as to coat surfaces of glass, paper, and cloth11,23,24 with CFPS devices. The protocols presented here demonstrate two methods for embedding CFPS reactions in macro-scale (i.e., &#62;1 mm) hydrogel matrices, using agarose as the exemplar material. Agarose was chosen due to its high water absorption capacity, controlled self-gelling properties, and tuneable mechanical properties11,24,25,26. Agarose also supports functional CFPS, is cheaper than many other hydrogel alternatives, and is biodegradable, making it an attractive choice as an experimental model system. These methods have, however, been previously demonstrated as appropriate for embedding CFPS in a range of alternative hydrogels11. Considering the wide range of applications of hydrogels and the functionality of CFPS, the methods demonstrated here can provide a basis from which researchers are able to develop biologically enhanced hydrogel materials suited to their own ends.
In previous studies, microgel systems with a size range of 1 &#181;m to 400 &#181;m have been used to perform CFPS in hydrogels submerged in reaction buffer23,27,28,29,30,31. The requirement to submerge hydrogels within CFPS reaction buffers, however, limits opportunities for their deployment as materials in their own right. The protocols presented here allow CFPS reactions to occur within hydrogels without the need to submerge the gels in reaction buffers. Second, the use of macroscale gels (between 2 mm and 10 mm in size) allows the study of the physical interaction between hydrogels and cell-free gene expression. For example, with this technique, it is possible to study how the hydrogel matrix affects CFPS reactions11 and how CFPS reactions can impact the hydrogel matrix31. Larger sizes of hydrogels also allow for the development of novel, bio-programmable materials32. Finally, by embedding CFPS reactions into hydrogels, there is also a potential reduction in the requirement for plastic reaction vessels. For the deployment of cell-free sensors, this has clear advantages over devices dependent on plasticware. Taken together, embedding CFPS reactions in hydrogels provides several advantages for the deployment of cell-free devices beyond the laboratory.
The overall goal of the methods presented here is to allow the operation of CFPS reactions within hydrogel matrices. Two different methods are demonstrated for embedding cell-free protein production reactions in macro-scale hydrogel materials (Figure 1). In Method A, CFPS components are introduced to lyophilized agarose hydrogels to form an active system. In Method B, molten agarose is mixed with CFPS reaction components to form a complete CFPS hydrogel system, which is then lyophilized and stored until needed. These systems can be rehydrated with a volume of water or buffer and analyte to commence the reaction.
This study uses Escherichia coli cell lysate-based systems. These are some of the most popular experimental CFPS systems, as E. coli cell lysate preparation is simple, inexpensive, and achieves high protein yields. The cell lysate is complemented with the macromolecular components needed to perform transcription and translation, including ribosomes, tRNAs, aminoacyl-tRNA synthetases, and initiation, elongation, and termination factors. Specifically, this paper demonstrates the production of eGFP and mCherry in agarose hydrogels using E. coli cell lysates and monitors the appearance of fluorescence using a plate reader and confocal microscopy. Representative results for the microtiter plate reader can be seen in Whitfield et al.31, and the underlying data are publicly available33. Further, the expression of fluorescent proteins throughout the gels is confirmed using confocal microscopy. The two protocols demonstrated in this paper allow for the assembly and storage of CFPS-based genetic devices in materia with the ultimate goal of creating a suitable physical environment for the distribution of cell-free gene circuitry in a manner supportive of field deployment.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-PGA</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-214793B</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10752.22</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Severn Biotech</td>
			<td>30-15-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Amino Acid Sampler Kit</td>
			<td>VWR</td>
			<td>BTRABR1401801</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A8937-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>Sigma-Aldrich</td>
			<td>A9501-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Coenzyme A (CoA)</td>
			<td>Sigma-Aldrich</td>
			<td>C4282-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>CTP</td>
			<td>Alfa Aesar</td>
			<td>J14121.MC</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0862</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>F7878-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP</td>
			<td>Carbosynth</td>
			<td>NG01208</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>K-glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G1149-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma-Aldrich</td>
			<td>L6876-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg-glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>49605-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>NAD</td>
			<td>Sigma-Aldrich</td>
			<td>N6522-250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-8000</td>
			<td>Promega</td>
			<td>V3011</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Hydroxide (KOH)</td>
			<td>Sigma-Aldrich</td>
			<td>757551-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic (K2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P3786-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>RDD037-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P2714-1BTL</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Protein concentration kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A50668</td>
			<td></td>
		</tr>
		<tr>
			<td>Rossetta 2 DE 3 E.coli</td>
			<td>Sigma-Aldrich</td>
			<td>71397-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma-Aldrich</td>
			<td>85558-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Thermo Fisher Scientific</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma-Aldrich</td>
			<td>GE17-1321-01</td>
			<td></td>
		</tr>
		<tr>
			<td>tRNA</td>
			<td>Sigma-Aldrich</td>
			<td>10109541001</td>
			<td></td>
		</tr>
		<tr>
			<td>UTP</td>
			<td>Alfa Aesar</td>
			<td>J23160.MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Sigma-Aldrich</td>
			<td>Y1625-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Sigma-Aldrich</td>
			<td>HS4323-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>10K MWCO dialysis cassettes</td>
			<td>Thermo Fisher Scientific</td>
			<td>66381</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Sarstedt</td>
			<td>62.554.502</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge bottles</td>
			<td>Sarstedt</td>
			<td>62.547.254</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL centrifuge bottles</td>
			<td>Thermo Fisher Scientific</td>
			<td>3120-9500</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha 1-2 LD Plus freeze-dryer</td>
			<td>Christ</td>
			<td>part no. 101521, 101522, 101527</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>H-X3R</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 384 well microtitre plates</td>
			<td>Fischer Scientific</td>
			<td>66</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvettes</td>
			<td>Thermo Fisher Scientific</td>
			<td>222S</td>
			<td></td>
		</tr>
		<tr>
			<td>Elga Purelab Chorus</td>
			<td>Elga</td>
			<td>#####</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Microcentrifuge 5425R</td>
			<td>Eppendorf</td>
			<td>EP00532</td>
			<td></td>
		</tr>
		<tr>
			<td>High Speed Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>B34183</td>
			<td></td>
		</tr>
		<tr>
			<td>JMP license</td>
			<td>SAS Institute</td>
			<td>15</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Stirrer</td>
			<td>Fischer Scientific</td>
			<td>15353518</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Amcor</td>
			<td>PM-966</td>
			<td></td>
		</tr>
		<tr>
			<td>Photospectrometer (Biophotometer)</td>
			<td>Eppendorf</td>
			<td>16713</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes and tips</td>
			<td>Gilson</td>
			<td>#####</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Balance</td>
			<td>Sartorius</td>
			<td>16384738</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 Fluorometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q32866</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>SHKE8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonic Dismembrator (Sonicator)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12893543</td>
			<td></td>
		</tr>
		<tr>
			<td>Static Incubator</td>
			<td>Sanyo</td>
			<td>MIR-162</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe and needles</td>
			<td>Thermo Fisher Scientific</td>
			<td>66490</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo max Q8000 (Shaking Incubator)</td>
			<td>Thermo Fisher Scientific</td>
			<td>SHKE8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Varioskan Lux platereader</td>
			<td>Thermo Fisher Scientific</td>
			<td>VLBL00GD1</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genie 2</td>
			<td>Cole-parmer</td>
			<td>OU-04724-05</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR PHenomenal pH 1100 L, ph/mv/&#176;c meter</td>
			<td>VWR</td>
			<td>662-1657</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for embedding cell-free protein synthesis reactions in macro-scale hydrogels

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. While the dominant paradigm for the deployment of gene networks is within a cellular chassis, synthetic gene networks may also be deployed in cell-free environments. Promising applications of cell-free gene networks include biosensors, as these devices have been demonstrated against biotic (Ebola, Zika, and SARS-CoV-2 viruses) and abiotic (heavy metals, sulfides, pesticides, and other organic contaminants) targets. Cell-free systems are typically deployed in liquid form within a reaction vessel. Being able to embed such reactions in a physical matrix, however, may facilitate their broader application in a wider set of environments. To this end, methods for embedding cell-free protein synthesis (CFPS) reactions in a variety of hydrogel matrices have been developed. One of the key properties of hydrogels conducive to this work is the high-water reconstitution capacity of hydrogel materials. Additionally, hydrogels possess physical and chemical characteristics that are functionally beneficial. Hydrogels can be freeze-dried for storage and rehydrated for use later. Two step-by-step protocols for the inclusion and assay of CFPS reactions in hydrogels are presented. First, a CFPS system can be incorporated into a hydrogel via rehydration with a cell lysate. The system within the hydrogel can then be induced or expressed constitutively for complete protein expression through the hydrogel. Second, cell lysate can be introduced to a hydrogel at the point of polymerization, and the entire system can be freeze-dried and rehydrated at a later point with an aqueous solution containing the inducer for the expression system encoded within the hydrogel. These methods have the potential to allow for cell-free gene networks that confer sensory capabilities to hydrogel materials, with the potential for deployment beyond the laboratory.

Author Spotlight: A Novel Approach for Embedding Cell-Free Protein Synthesis Reactions in Hydrogels 

Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels

We know living systems continuously integrate information from many different sources and that these signals trigger complex and subtle responses. Much of this information processing occurs at the molecular level. We're interested in operating these processes within materials, but without cells.The purpose is to give materials novel functionality. Challenges in this work are primarily associated with the cell-free protein synthesis reaction, rather than working with hydrogel. Cell reaction require high quality cell lysate as well as high quality and purity DNA to function well.This protocol allows researchers to embed molecular biology reactions into a physical biodegradable material that can be taken outside of the laboratory. Also, because the reaction output interacts directly with the material chassis, there is potential for the development of materials with novel sensing and response capabilities. In short-term, we see these methods as providing a novel route to the deployment of cell-free diagnostic devices.This protocol allows a number of devices to be created and spatially organized in a manner that is not possible for liquid reactions. Our future interests are twofold. First, we are interested in expanding the nature of the hydrogels we use to bring more material functionality to the work.Second, we are interested in increasing the complexity and performance of the gene networks we operate in gels, expanding the biological functionality of these systems.

This protocol details two methods for embedding CFPS reactions into hydrogel matrices, with Figure 1 presenting a schematic overview of the two approaches. Both methods are amenable to freeze-drying and long-term storage. Method A is the most utilized methodology for two reasons. First, it has been shown to be the most applicable method for working with a range of hydrogel materials11.&#160;Second, Method A allows for the parallel testing of genetic constructs. Method...

Watch this Scientific Journal Video about A Novel Approach for Embedding Cell-Free Protein Synthesis Reactions in Hydrogels at JoVE.com

Here, we present two protocols for embedding cell-free protein synthesis reactions in macro-scale hydrogel matrices without the need for an external liquid phase.

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. ...

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Research

JoVE Journal

Biology

Cell-Free Protein Synthesis in Rehydrated Lyophilized Hydrogels

To begin, transfer the hydrogels to 1.5 milliliter micro centrifuge tubes, trimming the gels with a scalpel if necessary to make them fit in the wells. In another 1.5 milliliter micro centrifuge tube, combine 10 microliters of the cell-free extract with 25 microliters of two x cell-free protein synthesis buffer and four micrograms of plasmid DNA. Make up to a total volume of 50 microliters with double distilled water to prepare the cell-free protein synthesis solution.Pipette the solution onto the freeze dried hydrogels, and allow the gels to soak in the cell-free protein synthesis system for five to 10 minutes at room temperature. Transfer the gels to a black 384 well microtiter plate using a spatula. Then, transfer the microtiter plate to a plate reader, and use the plate reader settings shown on the screen for fluorescence detection and analysis.

Cell-Free Protein Synthesis in Deployable Hydrogels

In a 1.5 milliliter microcentrifuge tube on ice, combine 10 microliters of the cell-free extract with four micrograms of plasmid DNA and 25 microliters of 2X cell-free protein synthesis buffer. Make up to a total volume of 50 microliters with double-distilled water to prepare the cell-free protein synthesis solution. Weigh 0.75 grams of agarose and add it to 100 milliliters of double-distilled water buffer to prepare 0.75%agarose.Microwave the 0.75%agarose in 30-second bursts at high power and pipette 50 microliters of the molten agarose into 1.5-milliliter microcentrifuge tubes or into molds of a desired shape. Place the molten agarose on a heat block set to 50 degrees Celsius and allow the agarose to cool but not polymerize. Then, mix the molten agarose with the cell-free protein synthesis solution by pipetting and stirring with the pipette tip.Allow the gels to cool to room temperature and polymerize for approximately two minutes. Transfer the polymerized agarose to 1.5-milliliter microcentrifuge tubes with a spatula and flash freeze in liquid nitrogen. Place the flash-frozen hydrogels into minus 80 degrees Celsius storage for one hour.After that, remove the microcentrifuge tube lids. Cover the tubes with a wax film and pierce the film to allow the moisture to be dried off. Set the temperature of the freeze dryer to minus 20 degrees Celsius and pressure to 0.1 millibars and freeze-dry the cell-free protein synthesis devices for 18 hours or overnight.Rehydrate the freeze-dried devices for 30 minutes with 50 microliters of double-distilled water without excess liquid, and transfer the gels to a black 384-well microtiter plate using a spatula. Finally, place the microtiter plate on a plate reader and use the plate reader settings shown on the screen for fluorescence detection and analysis. The cell-free protein synthesis of eGFP and mCherry in a hydrogel using E.coli cell lysates is shown in this figure.0.75%agarose gels were prepared without DNA template with four micrograms of either eGFP or mCherry template or with four micrograms of both eGFP and mCherry template. An overlay of the two channels is also shown, and the overlay includes the differential interference contrast image. The results confirmed the co-expression of both mCherry and eGFP in agarose.

838 Views.  Newcastle University.  Here, we present two protocols for embedding cell-free protein synthesis reactions in macro-scale hydrogel matrices without the need for an external liquid phase.

Embedding CFPS Reactions in a Variety of Hydrogel Matrices

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