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.

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

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

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.

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. ...

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1.1K 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.

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

Maintaining Wolbachia in Cell-free Medium

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ascertain the viability of the purified Wolbachia and (3) maintain the now extracellular Wolbachia in cell-free medium.  Purified Wolbachia remain alive in the extracellular phase but do not replicate until re-inoculated into eukaryotic cells.  Extracellular Wolbachia purified in this manner will remain viable for at least a week at room temperature, and possibly longer.  Purified Wolbachia are suitable for micro-injection, DNA extraction and other applications. 


This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...