The authors would like to acknowledge Dr. David Tirrell (Caltech) for his kind gift of the plasmid pQE9 AC10Atrp12, Dr. Tom Muir (Princeton University) for his kind gift of the plasmid KanR-IntRBS-NpuNC-CFN11, Dr. Takehisa Matsuda (Kanazawa Institute of Technology, Hakusan, Ishikawa, Japan) for his kind gift of the plasmid pET30-CutA-Tip110, and Dr. Jay D. Keasling (UC Berkley) for his kind gift of the plasmid pJD75713. This work was supported in part by the National Science Foundation CAREER, US Air force YIP and Norman Hackman Advanced Research Program.

1. Plasmid Construction
NOTE: All genes were amplified under standard PCR reactions using Phusion High-Fidelity DNA Polymerase per the manufacturer&#39;s specifications. Primers used for cloning have been described previously9. All constructs are listed in Table 1.
<ol>
	<li>To generate CutA-NpuN (N, Table 1):
	<ol>
		<li>PCR amplify CutA and NpuN genes from plasmids pET30-CutA-Tip110 and KanR-IntRBS-NpuNC-CFN11, respectively, using the appropriate primers.</li>
		<li>Digest these fragments with the appropriate restriction enzymes and sequentially insert these fragments into the pET26b vector between the T7 promoter and a C-terminal 6xHistidine tag to generate N (Figure 2A).</li>
	</ol>
	</li>
	<li>To generate NpuC-S-CutA (C, Table 1):
	<ol>
		<li>PCR amplify NpuC, CutA and S fragment [AG3(PEG)]10 from plasmid KanR-IntRBS-NpuNC-CFN11, pET30-CutA-Tip110 and pQE9 AC10Atrp12, respectively, using the appropriate primers.</li>
		<li>Digest these fragments with the appropriate restriction enzymes and sequentially insert these fragments into the pET26b vector between T7 promoter and a C-terminal 6xHistidine to generate C (Figure 2B).</li>
	</ol>
	</li>
	<li>To generate NpuC-S-SH3lig-CutA (C-SH3lig, Table 1):
	<ol>
		<li>PCR amplify CutA using primers containing a SH3lig (PPPALPPKRRR) and a flexible linker (GGGGS)2 to generate fragment SH3lig-CutA.</li>
		<li>Replace the CutA gene from C with fragment SH3lig-CutA.</li>
	</ol>
	</li>
	<li>To generate SH3-GFP (Table 1):
	<ol>
		<li>Amplify the SH3 gene from plasmid pJD75713 using the appropriate primers.</li>
		<li>Fuse this fragment to the GFP gene and insert it into the pET26b vector between the T7 promoter (Figure 2C) and a C-terminal 6xHistidine tag.</li>
	</ol>
	</li>
</ol>
2. Protein Expression
<ol>
	<li>Transform 50 &#956;l of chemically competent Escherichia coli BL21(DE3) with the appropriate expression plasmid.</li>
	<li>After transformation, serially dilute these cells, and plate them on Luria-Bertani (LB)/agar plates containing 50 &#956;g/ml&#160;kanamycin.</li>
	<li>Incubate plates containing transformed cells at 37 &#176;C for ~15 hr.</li>
	<li>After incubation, pick a plate that contains 50-100 colonies and resuspend all colonies in 5 ml&#160;of LB broth.</li>
	<li>Transfer suspension to 1 L LB broth containing kanamycin (50 &#956;g/ml) and grow cells at 37 &#176;C with shaking at 250 rpm. Monitor the absorbance at 600 nm (OD600). Grow culture until OD600 ~0.8.
	<ol>
		<li>For C and C-SH3lig, induce protein expression by adding isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) to the culture (1 mM final concentration) and incubate the culture at 37 &#176;C for 4 hr while shaking at 250 rpm.</li>
		<li>For N and SH3-GFP, cool the culture to ~18 &#176;C by immersing the culture flask in an ice water bath for ~5 min. Induce protein expression by adding IPTG to the culture (1mM final concentration) and incubate the culture at 18 &#176;C for 14-18 hr while shaking at 250 rpm.</li>
	</ol>
	</li>
	<li>After protein expression, centrifuge the culture at 6,000 x g for 20 min at 4 &#176;C to collect the pellet. Store cell pellet at -80 &#176;C until use.</li>
</ol>
3. Protein Purification
<ol>
	<li>Purification of N (denaturing conditions)
	<ol>
		<li>Resuspend cell pellets in Buffer A (Table 2) at 10 ml/g of wet pellet.</li>
		<li>Immerse the pellet suspension in an ice-water bath and disrupt cells by sonication (Amp 10, with 1 sec pulse and 6 sec pause for 1 min).</li>
		<li>Centrifuge the lysate at 16,000 x g for 20 min at 4 &#176;C.</li>
		<li>Discard the supernatant. Resuspend the pellet in Buffer DA (containing 8 M urea) and centrifuge the suspension at 16,000 x g for 20 min at 4 &#176;C.</li>
		<li>Pass the supernatant through a 5&#160;ml&#160;Ni-nitrilotriacetic acid (NTA) column previously equilibrated with buffer DA.</li>
		<li>Wash column with 30 ml&#160;of Buffer DA supplemented with 45 mM imidazole. Elute purified protein using 20 ml&#160;of Buffer DA supplemented with 150 mM imidazole.</li>
		<li>Reduce the urea concentration in the protein sample to &#60;1 mM by either one of the following methods given in 3.1.7.1 or 3.1.7.2:
		<ol>
			<li>Dialyze protein in DPBS buffer (Table 2) at 4 &#176;C overnight using tubes with &#60;20 kDa cutoff.</li>
			<li>Centrifuge purified protein in a 30&#160;kDa ultra-filtration spin column at 2,800 x g, 4 &#176;C until the volume is less than 1 ml. Add 14 ml&#160;DPBS buffer to the column to dilute the protein sample. Repeat the centrifugation/dilution steps three more times.</li>
		</ol>
		</li>
		<li>After buffer exchange, add dithiothreitol (DTT) to the purified protein (final 2 mM) and concentrate protein to ~100 mg/ml&#160;by centrifugation through a 30&#160;kDa ultra-filtration spin column at 2,800 x g, 4 &#176;C.</li>
		<li>Aliquot the concentrated protein and store at -80 &#176;C until use.</li>
	</ol>
	</li>
	<li>Purification of C and C-SH3lig (native condition)
	<ol>
		<li>Resuspend cell pellets in Buffer B (pH 6.0) (Table 2) supplemented with 1x protease inhibitor cocktail at 10 ml/g of wet pellet. Use acidic buffer to minimize proteolytic degradation of the target protein.</li>
		<li>Disrupt cell suspension by sonication as described in 3.1.2. Centrifuge the lysate at 16,000 x g for 20 min at 4 &#176;C and keep the supernatant.</li>
		<li>Pass the soluble lysate through a 5-ml&#160;Ni-NTA column previously equilibrated with buffer B.</li>
		<li>Wash column with Buffer B supplemented with 45 mM imidazole, and elute the target protein in 20 ml&#160;of Buffer B supplemented with 150 mM imidazole.</li>
		<li>For C, skip to step 3.2.6. For C-SH3lig, carry out an additional ion-exchange purification step to remove partially degraded protein as given in steps 3.2.5.1 to 3.2.5.3
		<ol>
			<li>Reduce NaCl concentration in C-SH3lig to &#60;1 mM following the procedure described in 3.1.7.</li>
			<li>Load the target protein onto a 5&#160;ml&#160;anion exchanger beaded agarose matrix column previously equilibrated with sodium phosphate buffer (50 mM, pH 7.0).</li>
			<li>Elute target protein from the column by running a gradient from a solution containing 10 mM Tris-HCl pH 8.0 buffer to a solution containing the same buffer supplemented with 1 M NaCl. Take samples during protein elution and pool samples with the highest purity based on sodium dodecyl sulfate&#160;polyacrylamide gel electrophoresis (SDS-PAGE).</li>
		</ol>
		</li>
		<li>Buffer exchange the purified protein into DPBS buffer, as described in 3.1.7.</li>
		<li>Add DTT to the purified protein (final concentration 2 mM) and concentrate the protein to ~100 mg/ml&#160;using a 30&#160;kDa ultra-filtration spin column as described in 3.1.8. Aliquot and store concentrated protein at -80 &#176;C until use.</li>
	</ol>
	</li>
	<li>Purification of SH3-GFP
	<ol>
		<li>Resuspend cell pellets using Buffer A at 10 ml/g of wet pellet.</li>
		<li>Disrupt pellet suspension by sonication as described in 3.1.2.</li>
		<li>Centrifuge the lysate at 16,000 x g for 20 min at 4 &#176;C and collect the supernatant.</li>
		<li>Pass the supernatant (soluble lysate) through a 5-ml&#160;Ni-NTA column previously equilibrated with Buffer A.</li>
		<li>Wash column with 30 ml&#160;of Buffer A supplemented with 45 mM imidazole. Elute purified protein using 20 ml&#160;of Buffer A supplemented with 150 mM imidazole.</li>
		<li>Buffer exchange the purified protein into DPBS buffer using an approach similar to that described in 3.1.7 and concentrate the protein to ~150 mg/ml&#160;using a 30&#160;kDa ultra-filtration spin column as described in 3.1.8.</li>
		<li>Aliquot and store purified protein at -80 &#176;C until use.</li>
	</ol>
	</li>
	<li>SDS-PAGE analysis of purified samples containing CutA
	<ol>
		<li>Dilute each purified protein in double-distilled water to reduce the concentration of NaCl to ~1 mM. At this NaCl concentration, most of the CutA trimer proteins run as monomers on the SDS-PAGE gels.</li>
		<li>Mix samples with 2x SDS sample buffer (0.5 M Tris-HCl, pH 6.8, 20% Glycerol, 10% w/v SDS, 0.1 % w/v bromo-phenol blue, 2% &#946;-mercaptoethanol), incubated at 95 &#176;C for 5 min.</li>
		<li>Load the samples onto a 12% SDS-PAGE gel. Carry out electrophoresis at a constant voltage of 200 V for ~50 min.</li>
		<li>Observe protein in the gels by staining with Coomassie brilliant blue R250 following the standard protocols (Figure 1C).</li>
	</ol>
	</li>
</ol>
4. Hydrogel Formation
NOTE: the sample hydrogels made in this study contain 1.6 mM of each hydrogel building block unless noted otherwise. This protein concentration yields a soft and stable hydrogel. CAUTION: Sodium azide (NaN3) is added to the hydrogel to a final concentration of 0.5% w/v to prevent bacterial contamination. NaN3 is highly toxic and must be handled with extreme care as indicated in the Material Safety Data Sheet.
<ol>
	<li>Calculate the volume for each of the concentrated proteins needed to achieve a final concentration of 1.6 mM in a 100 &#956;l&#160;sample hydrogel. For example: 
	Concentration of N: 100 mg/ml 
	Molecular weight of N: 26.3 kDa (refer to Table 1) 
	Desired Volume: 100 &#956;l Desired Concentration: 1.6 mM 
	<img alt="" fo:content-width="4.5in" fo:src="/files/ftp_upload/51202/book.JPG" src="/files/ftp_upload/51202/book.JPG" width="600" /></li>
	<li>To make a 100 &#956;l&#160;hydrogel (1.6 mM), mix C (x &#956;l, volume calculated according to 4.1) with 5% NaN3 (10 &#956;l), 100 mM DTT (5 &#956;l) and N (y&#160;&#956;l, volume calculated according to 4.1) inside a 2 ml&#160;glass vial.</li>
	<li>Add DPBS buffer ((85 -&#160;x - y) &#956;l) to the vial to achieve a final volume of 100 &#956;l, and manually mix all the components via a swirling motion using a pipette tip. Note: The solution becomes very viscous upon mixing.</li>
	<li>Centrifuge the mixture for 2 min at 8,000 x g to remove the air bubbles.</li>
	<li>Incubate the mixture at room temperature overnight to allow the intein trans-splicing reaction to reach completion. Confirm hydrogel formation by turning tube upside down. The proteins will not flow if a hydrogel is formed.</li>
	<li>Estimate the intein trans-splicing yield by checking samples (0.5 &#956;l&#160;each) collected before step 4.2 and after step 4.5 on a SDS-PAGE gel, as described in 3.4 (Figure 1C).</li>
</ol>
5. Immobilization of GFP via Docking Protein (DP) and Docking Station Peptide (DSP) Interaction
<ol>
	<li>To make a 50 &#956;l&#160;GFP-functionalized hydrogel (1.2 mM), combine C-SH3lig (x &#956;l, calculated according to 4.1) and SH3-GFP (y &#956;l, calculated according to 4.1) at 1:1 molar ratio in a 1.7 ml&#160;microcentrifuge tube and incubate the mixture at room temperature for 30 min.</li>
	<li>Add 5% NaN3 (5 &#956;l), 100 mM DTT (2.5 &#956;l), (42.5 -&#160;x&#160;-&#160;y) &#956;l DPBS to the same tube. Add N (y &#956;l, calculated according to 4.1) to achieve a 1:1 molar ratio of N and C-SH3lig. Mix the sample by using a pipette tip by a swirling motion.</li>
	<li>Centrifuge the mixture at 8,000 x g for 2 min and incubate the mixture at room temperature overnight in the dark. A hydrogel encapsulating SH3-GFP forms during incubation.</li>
</ol>
6. Use of 1.6 mM Hydrogel as an Immobilization Scaffold for Enzymatic Reaction in Organic Solvent
<ol>
	<li>Use the HRP as a model enzyme. Prepare a stock solution of HRP (28 mg/ml&#160;or 0.63 mM) in DPBS.</li>
	<li>To make a 30 &#956;l&#160;hydrogel (1.6 mM) entrapping HRP, combine C (x &#956;l, calculated according to 4.1) with HRP (2 &#956;l), 5% NaN3 (3 &#956;l) and DTT (1.5 &#956;l&#160;of 100 mM) inside a 1.7 ml&#160;centrifuge tube.</li>
	<li>Add N (y &#956;l, calculated according to 4.1) and DPBS (23.5 -&#160;x&#160;-&#160;y) &#956;l. Mix with a pipette tip with a swirling motion.</li>
	<li>Centrifuge the mixture at 8,000 x g for 2 min and incubate at room temperature overnight. 
	CAUTION: the regents used for the following activity assay are highly toxic. Use specific safety recommendations by the corresponding Material Safety Data Sheets.</li>
	<li>For enzymatic reaction, submerge the hydrogel in 1 ml&#160;of reaction cocktail containing N,N-dimethyl-p-phenylene diamine (5.8 mM), phenol (5.8 mM) and tert-butyl hydroperoxide (2.9 mM) in n-heptane14. Manually disrupt the gel using a pipette tip to increase the contact surface area of the hydrogel and the solvent.</li>
	<li>Detect HRP product, an indophenol-type dye, by measuring the optical absorbance of samples taken at different times at 546 nm in a plate reader (Figure 5).</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+naturally+split+Npu+DnaE+intein+exhibits+an+extraordinarily+high+rate+in+the+protein+trans-splicing+reaction.">The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction.</a> FEBS Lett. 583, 909-914 (2009).</li><li>Tanaka, Y., 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=Structural+implications+for+heavy+metal-induced+reversible+assembly+and+aggregation+of+a+protein:+the+case+of+Pyrococcus+horikoshii+CutA.">Structural implications for heavy metal-induced reversible assembly and aggregation of a protein: the case of Pyrococcus horikoshii CutA.</a> FEBS Lett. 556, 167-174 (2004).</li><li>Sawano, 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=Thermodynamic+basis+for+the+stabilities+of+three+CutA1s+from+Pyrococcus+horikoshii,Thermus+thermophilus,+and+Oryza+sativa,+with+unusually+high+denaturation+temperatures.">Thermodynamic basis for the stabilities of three CutA1s from Pyrococcus horikoshii,Thermus thermophilus, and Oryza sativa, with unusually high denaturation temperatures.</a> Biochemistry. 47, 721-730 (2008).</li><li>Tanaka, 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=Hyper-thermostability+of+CutA1+protein,+with+a+denaturation+temperature+of+nearly+150+degrees+C..">Hyper-thermostability of CutA1 protein, with a denaturation temperature of nearly 150 degrees C..</a> FEBS Lett. 580, 4224-4230 (2006).</li><li>Shen, W., Zhang, K., Kornfield, J. 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No competing financial interests exist.

In this work, we demonstrated the synthesis of a highly stable intein-mediated protein hydrogel. The use of a split intein enables the hydrogel to be conditionally formed in response to the mixing of two liquid-phase components. Specifically, the split intein covalently links two liquid-phase building blocks via a trans-splicing reaction, yielding a polypeptide unit flanked by crosslinking units that in turn self assembles into a hydrogel. The mixing-induced formation of the hydrogel bypasses technical difficult...

Hydrogels made entirely of proteins carry the potential to significantly advance fields as diverse as tissue engineering, drug delivery and biofabrication1. They offer advantages over traditional synthetic polymer hydrogels including biocompatibility and the potential to noninvasively support the incorporation of bioactive globular proteins.
In this work, we describe the development of a novel protein hydrogel formed via a split-intein-mediated protein trans-splicing reaction and its application as a protein immobilization scaffold (Figure 1). The building blocks for this hydrogel are two protein block-copolymers each comprising the N- or C-terminal fragment of a split intein (IN and IC) and a subunit of a multimeric crosslinker protein. The DnaE intein from Nostoc punctiforme (Npu) was used as the split intein2,3 and a small trimeric protein (12 kDa) CutA from Pyrococcus horikoshii was used as the crosslinker protein4,5. Different crosslinkers are joined through intein catalyzed trans-splicing reaction, leading to the formation of a highly crosslinked protein network (hydrogel). Npu intein was chosen because of its fast reaction kinetics (t1/2 = 63 sec) and high trans-splicing yield (close to 80%)2,3. The CutA protein was chosen as the crosslinker due to its high stability. CutA trimers have a denaturation temperature of near 150 &#176;C and retain trimeric quaternary structure in solutions containing as much as 5 M guanidine hydrochloride 4,6. Since subunit exchange between different crosslinkers is a major contributor of the physical hydrogel surface erosion7, the very strong inter subunit interaction in CutA should discourage such subunit exchanges, leading to a more stable hydrogel. One of these building blocks also contains a highly hydrophilic peptide S-fragment as the mid-block to facilitate water retention8.
Mixing of the two hydrogel building blocks initiates a trans-splicing reaction between the IN and IC intein fragments, generating a longer polypeptide chain with crosslinkers at both terminals. Crosslinkers from multiple such molecular units interact with each other, forming a highly crosslinked hydrogel network (Figure 1A). A specific &#34;docking station peptide&#34; (DSP) is incorporated into one of the hydrogel building blocks to facilitate stable immobilization of a &#34;docking protein&#34; (DP)-tagged target protein into the hydrogel. The use of a split intein to mediate the hydrogel assembly not only provides additional flexibility for protein hydrogel synthesis, but also enables high-density, uniform loading of the target protein throughout the entire hydrogel, as the target proteins are loaded prior to hydrogel formation.
The intein-mediated protein hydrogel is highly stable in aqueous solution with little-to-no detectable erosion after 3 months at room temperature. Stability is retained in a wide range of pHs (6-10) and temperatures (4-50 &#176;C), and the hydrogel is also compatible with organic solvents. This hydrogel is used for the immobilization of two globular proteins: the green fluorescent protein (GFP) and the horseradish peroxidase (HRP). Hydrogel entrapping the latter protein is used to perform biocatalysis in an organic solvent.

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			Name 
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			Company
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			Catalog Number
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			Comments
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			Phusion High Fidelity DNA polymerase
			</td>
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			New England BioLabs
			</td>
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			M0530S
			</td>
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		<tr>
			<td style="width:175px;height:21px;">
			Competent Escherichia coli BL21 (DE3)
			</td>
			<td nowrap="nowrap" style="width:111px;height:21px;">
			New England BioLabs
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			C2527I
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:175px;height:21px;">
			Luria Bertani
			</td>
			<td style="width:111px;height:21px;">
			VWR
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			90003-350
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:175px;height:21px;">
			Bacto Agar Media
			</td>
			<td style="width:111px;height:21px;">
			VWR
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			<a href="https://us.vwr.com/store/catalog/product.jsp?catalog_number=90000-760">90000-760</a>
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:175px;height:21px;">
			kanamycin sulfate
			</td>
			<td nowrap="nowrap" style="width:111px;height:21px;">
			VWR
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			<a href="https://www.vwrsp.com/catalog/product/index.cgi?catalog_number=97061-602">97061-602</a>
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:175px;height:21px;">
			IPTG
			</td>
			<td nowrap="nowrap" style="width:111px;height:21px;">
			VWR
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			EM-5820
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:175px;height:21px;">
			Imidazole
			</td>
			<td nowrap="nowrap" style="width:111px;height:21px;">
			VWR
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			EM-5720
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:175px;height:21px;">
			Urea
			</td>
			<td nowrap="nowrap" style="width:111px;height:21px;">
			VWR
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			EM-9510
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:175px;height:21px;">
			Dithiothreitol (DTT)
			</td>
			<td nowrap="nowrap" style="width:111px;height:21px;">
			Fisher
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			BP172-5
			</td>
			<td style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:175px;height:21px;">
			Protease Inhibitor cocktail
			</td>
			<td nowrap="nowrap" style="width:111px;height:21px;">
			Roche Applied Science
			</td>
			<td style="width:118px;height:21px;">
			11836153001
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:175px;height:21px;">
			DPBS
			</td>
			<td style="width:111px;height:21px;">
			VWR
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			82020-066
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:175px;height:21px;">
			Brilliant Blue R
			</td>
			<td style="width:111px;height:21px;">
			Acros Organics
			</td>
			<td style="width:118px;height:21px;">
			A0297990
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:175px;height:21px;">
			Sodium Azide
			</td>
			<td nowrap="nowrap" style="width:111px;height:21px;">
			Fisher
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			AC190380050
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			Caution, highly toxic
			</td>
		</tr>
		<tr>
			<td style="width:175px;height:21px;">
			Horseradish peroxidase
			</td>
			<td style="width:111px;height:21px;">
			Sigma
			</td>
			<td style="width:118px;height:21px;">
			P8125-5KU
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:175px;height:21px;">
			N,N-dimethyl-p-phenylene diamine
			</td>
			<td style="width:111px;height:21px;">
			Fisher
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			AC408460250
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			Caution, highly toxic
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:175px;height:21px;">
			phenol
			</td>
			<td style="width:111px;height:21px;">
			Fisher
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			AC149340500
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			Caution, highly toxic
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:175px;height:21px;">
			tert-butyl hydroperoxide
			</td>
			<td style="width:111px;height:21px;">
			Fisher
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			AC180340050
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			Caution, highly toxic
			</td>
		</tr>
		<tr>
			<td style="width:175px;height:21px;">
			n-heptane
			</td>
			<td nowrap="nowrap" style="width:111px;height:21px;">
			Acros Organics
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			120340010
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:175px;height:21px;"></td>
			<td style="width:111px;height:21px;"></td>
			<td style="width:118px;height:21px;"></td>
			<td nowrap="nowrap" style="width:137px;height:21px;">[header]</td>
		</tr>
		<tr>
			<td style="width:175px;height:21px;">
			Shaker/Incubator
			</td>
			<td style="width:111px;height:21px;">
			Fisher Scientific
			</td>
			<td style="width:118px;height:21px;">
			Max Q 6000
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:175px;height:21px;">
			Centrifuge
			</td>
			<td style="width:111px;height:21px;">
			Sorvall
			</td>
			<td style="width:118px;height:21px;">
			RC 6
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:175px;height:21px;">
			Sonicator
			</td>
			<td nowrap="nowrap" style="width:111px;height:21px;">
			QSonica
			</td>
			<td style="width:118px;height:21px;">
			Misonix 200
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:175px;height:21px;">
			Ultrafiltration Tubes
			</td>
			<td style="width:111px;height:21px;">
			Amicon Ultra
			</td>
			<td style="width:118px;height:21px;">
			UFC903024
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:175px;height:21px;">
			&#160;Ni Sepharose High Performance HisTrap column
			</td>
			<td nowrap="nowrap" style="width:111px;height:21px;">
			GE Healthcare Life Sciences
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			17-5248-01
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:175px;height:21px;">
			HiTrap SP Sepharose FF ion exchange column
			</td>
			<td nowrap="nowrap" style="width:111px;height:21px;">
			GE Healthcare Life Sciences
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			17-5156-01
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:175px;height:21px;">
			Plate reader
			</td>
			<td nowrap="nowrap" style="width:111px;height:21px;">
			Molecular Devices
			</td>
			<td nowrap="nowrap" style="width:118px;height:21px;">
			SpectraMax Gemini EM
			</td>
			<td nowrap="nowrap" style="width:137px;height:21px;">
			
			</td>
		</tr>
	</tbody>
</table>

synthesis of an intein-mediated artificial protein hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

A schematic for intein-mediated protein hydrogel formation is presented in Figure 1A. The building blocks of the hydrogel are the protein copolymers CutA-NpuN (N) and NpuC-S-CutA(C) (Figure 1A, Table 1). NpuN/C are the N-/C-fragments of the naturally split DnaE intein from Nostoc punctiforme (Npu). CutA is a stable trimeric protein from Pyrococcus horikoshii4,5. Mixing of purified N and C in the presence of the reducing agent DTT induces the formation...

Watch this Scientific Journal Video about Synthesis of an Intein-mediated Artificial Protein Hydrogel at JoVE.com

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

synthesis-of-an-intein-mediated-artificial-protein-hydrogel

Research

JoVE Journal

Bioengineering

11.9K Views.  Texas A&M University, College Station. We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic so...

Video: Synthesis of an Intein-mediated Artificial Protein Hydrogel

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

An Additive Manufacturing Technique for the Facile and Rapid Fabrication of Hydrogel-based Micromachines with Magnetically Responsive Components

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels have simple monolithic architectures and often function as scaffolding materials for tissue engineering applications. More sophisticated structures typically take a long time to fabricate and do not contain moving components. This protocol describes a photolithography method that allows for facile and rapid microfabrication of PEG structures and devices. This strategy involves an in-house developed fabrication stage that allows for the rapid fabrication of 3D structures by building upwards in a layer-by-layer fashion. Independent moving components can also be aligned and assembled onto support structures to form integrated devices. These independent components are doped with superparamagnetic iron oxide nanoparticles that are sensitive to magnetic actuation. In this manner, the fabricated devices can be actuated using external magnets to yield movement of the components within. Hence, this technique allows for the fabrication of sophisticated MEMS-like devices (micromachines) that are composed entirely out of a biocompatible hydrogel, able to function without an onboard power source, and respond to a contact-less method of actuation. This manuscript describes the fabrication of both the fabrication set-up as well as the step-by-step method for the microfabrication of these hydrogels-based MEMS-like devices.

An additive manufacturing strategy for processing UV-crosslinkable hydrogels has been developed. This strategy allows for the layer-by-layer assembly of microfabricated hydrogel structures as well as the assembly of independent components, yielding integrated devices containing moving components that are responsive to magnetic actuation.

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

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

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.