The authors acknowledge NIH 1R35GM138265-01 and NSF 2029532 for partial support of this project.

1. Designing the gene fragment
NOTE: The gene fragment should have all the necessary genetic elements for transcription/translation, including promoter, ribosome binding site (RBS), start codon, the gene of interest, and terminator. While the terminator is not necessary for a linear expression template (LET), it will be important if the user decides to insert the sequence into a plasmid. These sequences were lifted from the pJL1-sfGFP plasmid55 (gift from Michael Jewett&#39;s lab), which uses a T7 promoter. In addition to these necessary genetic elements, a restriction enzyme cut site is added six base pairs before the promoter (5&#39; cut site) and another six base pairs after the terminator (3&#39; cut site), in this case using HindIII (other restriction enzymes can be used, but it is helpful to standardize the sequences with one high fidelity restriction enzyme to reduce the number needed to keep in the library). Primer sites are added ten base pairs upstream of the 5&#39; cut site and ten base pairs downstream of the 3&#39; cut site, in this case using standardized M13 primer sequences (primers are inexpensive stock items). The restriction enzyme site and primers used are at the discretion of the user. However, the user must ensure the sequences are not present anywhere else in the template (do not want to create unwanted cuts or sites of amplification initiation). The sequences for the templates used in this work are detailed in the supplemental material. These steps are used to modify from this base template.
<ol>
	<li>Determine the desired gene to be expressed and obtain the amino acid sequence or the genetic sequence if it has been expressed in E. coli.</li>
	<li>If it is an amino acid sequence, perform codon optimization for E. coli using one of many standard vendor tools56. If using the template provided in the supplement, ensure the optimized sequence has no HindIII restriction sites (AAGCTT). In the case that it does, continue to optimize the sequence until there is no longer a HindIII site.</li>
	<li>Copy the sequence and paste it into the provided template for Supplementary Sequence #1 where the gene of interest is indicated. If expressing sfGFP, use Supplementary Sequence #1 as is. If expressing subtilisin, use Supplementary Sequence #2 as is.</li>
	<li>Order the minimal template and the necessary primers from the preferred DNA synthesis service.</li>
</ol>
2. Resuspending the gene fragment and the primers
NOTE: Upon receipt of the gene fragment, follow the manufacturer&#39;s protocols for resuspension or use this simple guide to create a DNA stock.
<ol>
	<li>Centrifuge the tube (300 x g for 5 s) to collect the DNA pellet at the bottom.</li>
	<li>Add double distilled water (ddH2O) to make a final concentration of 10 ng/&#181;L of DNA template.</li>
	<li>Vortex the solution on a medium setting for 5-10 s.</li>
	<li>Dissolve the entire pellet by incubating at 50 &#176;C for 20 min.</li>
	<li>Briefly vortex again</li>
	<li>Centrifuge at 300 x g for 5 s to collect the solution at the bottom of the tube.</li>
	<li>Store at -20 &#176;C or use in PCR.</li>
	<li>Prepare a 100 &#181;M primer stock by resuspending the primers in nuclease-free water. To determine the amount of water to add, multiply the number of nanomoles of lyophilized primer by 10. For example, if the tube contains 45 nM of lyophilized primer, add 450 &#181;L of ddH2O and vortex the solution.</li>
	<li>Store the primer stock solutions at -20 &#176;C or continue to perform the amplification.</li>
</ol>
3. Amplifying the gene fragment via PCR
NOTE: Decide which PCR kit is right for the gene of interest. Smaller genes (&#60;1,000 kb) may be more amenable to a cheaper Taq polymerase, while larger genes (&#8805;1,000 kb) may benefit from high fidelity polymerase to reduce errors. It is important to note that this initial PCR amplification is not necessary if the user is not concerned with preserving the initial gene fragment (It provides multiple attempts at circularization and allows for comparative studies of LET vs. RCA product). It is also important to note that this PCR amplified LET can be used directly in reactions; however, as mentioned in the introduction, it would only allow for a limited number of reactions if the further amplification steps were disregarded. Digestion and ligation can be performed on the resuspended gene fragment directly57 (if one is certain, they will not need more LET to perform additional circularization stocks). If this is the case, skip section 3 and continue to section 4. For performing PCR, follow these steps.
<ol>
	<li>Use the 100 &#181;M stocks from step 2.8 to create 10 &#181;M working solutions. Many PCR kit protocols call for 10 &#181;M solutions of primers.</li>
	<li>Program the thermal cycler to conduct the reaction according to the kit manufacturer&#39;s protocols. Different kits call for slightly varied cycling parameters. For the kit listed in the Table of Materials, the conditions are 94 &#176;C for 30 s of initial denaturation; 30 cycles of 94 &#176;C for 30 s of denaturing, 45 &#176;C for 30 s of primer annealing, and 68 &#176;C for 60 s of extension; with a final extension at 68 &#176;C for 5 min; and finally, a 10 &#176;C indefinite hold.
	<ol>
		<li>Ensure to select the correct elongation time (variable depending on the length of the gene to be amplified). Have an elongation time of 1 min for every 1,000 bp.</li>
		<li>Ensure to enter the correct annealing temperature for the primers. Use an online Tm calculator that uses both primers as inputs to determine the best annealing temperature58. An annealing temperature of 45 &#176;C is sufficient when using M13 primers.</li>
		<li>When determining the number of cycles, refer to the manufacturer&#39;s protocol, but 30 cycles will most often result in sufficient amplification.</li>
	</ol>
	</li>
	<li>If performing PCR, thaw and vortex the dNTPs. Use the PCR buffer provided in the kit.</li>
	<li>In a single PCR tube, combine all the kit components as directed in the manufacturer&#39;s protocol. To ensure successful amplification, add 1 &#181;L of resuspended DNA stock (step 2.6).</li>
	<li>Gently homogenize the mixture by vortexing on medium setting for 5-10 s. Alternatively, pipette half the volume up and down 10-20 times to vortex.</li>
	<li>Perform the PCR reaction.</li>
	<li>If the PCR protocol did not include a final cooling step, allow the reaction to cool for 5 min at 10 &#176;C before removing to drive condensation to the bottom of the tube.</li>
	<li>Purify the reaction using a PCR clean-up kit following the vendor&#39;s instructions.
	<ol>
		<li>In a 1.5 mL tube, add DNA binding buffer and PCR sample at a ratio of 5:1, respectively.</li>
		<li>Transfer this mixture to the spin column and centrifuge at 16,000 x g for 1 min. Discard the flow-through.</li>
		<li>Add 200 &#181;L of DNA wash buffer to the column and incubate at room temperature for 1 min.</li>
		<li>Centrifuge for 1 min at 16,000 x g and discard the flow-through.</li>
		<li>Repeat steps 2.8.3 and 2.8.4 without the 1 min incubation step.</li>
		<li>Centrifuge for an additional 1-2 min at 16,000 x g to remove any remaining buffer.</li>
		<li>Elute the DNA in 46 &#181;L of ddH2O.</li>
	</ol>
	</li>
	<li>Quantify the purified DNA using a spectrophotometer.</li>
	<li>Store the purified DNA at -20 &#176;C or proceed to the next step.</li>
</ol>
4. Digestion and circularization
NOTE: Further amplification can be achieved by circularizing the DNA followed by RCA. Digest the DNA to prepare the template for circularization. This will remove the primer sequences and create sticky ends at both the 5&#39; and 3&#39; ends of the template. Reattach these ends via ligation reaction.
<ol>
	<li>In a PCR tube, combine 5 &#181;L of the necessary buffer, 20 U of HindIII, and 45 &#181;L of the purified DNA from step 3.8.</li>
	<li>Gently homogenize this mixture with a pipette.</li>
	<li>Incubate the mixture in a thermal cycler for 15 min at 37 &#176;C.</li>
	<li>Heat inactivate HindIII by incubating for 20 min at 80 &#176;C.</li>
	<li>Allow the reaction to cool to 10 &#176;C before removing to drive condensation to the bottom of the tube.</li>
	<li>Add 5 &#181;L of T4 ligase buffer and 800 U of T4 ligase to the newly digested DNA.
	<ol>
		<li>Use T7 ligase, if desired.</li>
	</ol>
	</li>
	<li>Gently homogenize this mixture with a pipette.</li>
	<li>Incubate the mixture for 1 h at 25 &#176;C to perform the circularization reaction.</li>
	<li>Purify the reaction using a PCR clean-up kit following the vendor&#39;s instructions. Use the same protocol detailed in step 3.8.</li>
	<li>Quantify the DNA using a spectrophotometer. The expected values are ~20 ng/&#181;L.</li>
	<li>Store at -20 &#176;C or proceed to the next step.</li>
</ol>
5. Isothermal rolling circle amplification
NOTE: The Rolling circle amplification (RCA) can be performed using a commercial kit or with individually purchased components. Following the manufacturer&#39;s protocol will ensure a successful amplification. Kits typically contain a sample buffer, reaction buffer, and strand displacing polymerase, such as &#966;29 polymerase. Multiple reaction tubes can be combined to produce a large amount of DNA for cell-free expression (4 &#181;g from 20 pg of starting material). The following protocol works efficiently.
<ol>
	<li>In a single tube, combine 20 &#181;L of the sample buffer, 20 &#181;L of the reaction buffer, 0.8 &#181;L of the enzyme, and 1 &#181;L of the circular expression template (CET) from step 4.9. 
	NOTE: This will have a total DNA mass of ~20 ng, but RCA can work with picogram amounts, thus allowing the dilution of the CET and extreme enzymatic amplification if there is a significant need for material in the high throughput screening.</li>
	<li>Homogenize the mixture with a pipette and aliquot 10 &#181;L of the mixture into four separate tubes.</li>
	<li>Incubate at 30 &#176;C for 4-18 h.</li>
	<li>Heat inactivate the enzyme by incubating at 65 &#176;C for 10 min. Reduce the temperature to 12 &#176;C for 5 min to encourage condensation at the bottom of the tube. 
	NOTE: It&#39;s easiest to combine all temperature steps in an automated protocol on a thermal cycler.</li>
	<li>Dilute the resulting solution by adding 15 &#181;L of ddH2O to each tube.</li>
	<li>Combine all tubes and add directly to a cell-free reaction.</li>
	<li>If desired, use a PCR clean-up kit to purify the product and elute it in 36 &#181;L of ddH2O to quantify. Ensure that the template concentration is ~100 ng/&#181;L.</li>
</ol>
6. Cell-free reaction
NOTE: Perform cell-free expression by combining energy buffer, extract, and RCA template. A typical cell-free reaction using the PANOx-SP energy buffer consists of 1.2 mM ATP, 0.85 mM each of GMP, UMP, and CMP, 30 mM phosphoenolpyruvate, 130 mM potassium glutamate, 10 mM ammonium glutamate, 12 mM magnesium glutamate, 1.5 mM spermidine, 1 mM putrescine, 34 &#181;g/mL of folinic acid, 171 &#181;g/mL of E. coli tRNA mixture, 2 mM each of 20 unlabeled amino acids, 0.33 mM NAD, 0.27 mM Coenzyme A (CoA), 4 mM potassium oxalate, 57 mM HEPES-KOH buffer (pH 7.5), 0.24% volume of the E. coli extract, and variable amounts of DNA23,49. The volume of reaction can vary but 15 &#181;L reactions can save on reagent usage and are small enough for use in a 384 black-walled microplate49,50.
<ol>
	<li>If expressing a fluorescent protein such as sfGFP, prepare a plate reader to read at the desired excitation/emission, temperature, and agitation.</li>
	<li>If using a 384-well plate, aliquot 60 &#181;L of H2O into the wells bordering an empty sample well to maintain humidity and reduce the edge effect.</li>
	<li>Add the various required components into a tube for each sample. Add enough to perform triplicates. Replicates within the plate can help identify causes of variability.
	<ol>
		<li>Add the extract, the energy buffer and then the DNA.</li>
		<li>Dilute to the final desired volume with ddH2O.</li>
	</ol>
	</li>
	<li>Mix this solution thoroughly by pipetting half of the solution volume up and down 10-20 times.</li>
	<li>Transfer the reaction mixture in 15 &#181;L aliquots to the desired wells in the microtiter plate.</li>
	<li>Seal the plate with a colorless sealing film to maintain humidity and prevent evaporation.</li>
	<li>Place the sealed plate in the plate reader and allow the reaction to complete.
	<ol>
		<li>If expressing a protein that does not have the capability of being monitored live, use another temperature-controlled apparatus such as a thermoblock to incubate the plate.</li>
	</ol>
	</li>
</ol>
7. Subtilisin assay
NOTE: If expressing the subtilisin BPN&#39; (SBT(n)) gene in Supplementary Sequence #2, follow this protocol to assay the activity.
<ol>
	<li>Prepare a 10 &#181;M stock solution of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide in dimethylformamide (DMF).</li>
	<li>Set a plate reader to measure absorbance at 410 nm every 20 s for 10 min while maintaining a temperature of 25 &#176;C.</li>
	<li>In a flat bottom, colorless 96-well plate, aliquot 94 &#181;L of ddH2O and 1 &#181;L of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide from step 7.1.</li>
	<li>Add 5 &#181;L of the finished cell-free reaction from step 6.7 and read using a plate reader set to the protocol described in step 7.2.</li>
</ol>

Nigel Reuel serves on the scientific advisory board of BigHat Biosciences Inc., a company that uses cell-free systems for the design of antibodies.

The gene of interest can be any desired protein, but it is best to start with a fluorescent protein as a convenient reporter for real-time or end-point readout on a well plate reader for new adopters of this method. For new protein sequences, copy the amino acid sequence of the desired protein and paste it into the desired codon optimization tool61,62. There are usually many available organisms and strains of E. coli in the codon optimization tool, but c...

Cell-free gene expression (CFE) has emerged as a powerful tool with many applications. Such applications include disease detection1,2,3,4,5,6, micronutrient and small molecule detection7,8,9,10,11,12, biomanufacturing13,14,15,16,17,18, education19,20,21, manufacturing difficult proteins17,22,23,24,25,26,27, and variant screening23,28,29,30,31,32,33. This is due to the open nature of cell-free systems and the flexibility they confer. Many great review articles offer historical education and future perspectives on the technology34,35,36,37,38,39,40,41,42,43,44.
A typical cell-free reaction consists of three major components: cell extract, energy mix, and genetic template. Active cell extract contains all the necessary machinery for transcription and translation (TXTL) and can be processed in a variety of ways36. Glycolytic intermediates, electrolytes, amino acids, and cofactors in the energy mix support the TXTL process. It is a major source of variability in cell-free experiments45 and can be prepared in many ways34,46. Preparation of the genetic template has seen fewer improvements since traditional cloning methods result in plasmids with excellent expression characteristics. The downside to these traditional methods is the turnaround time and amount of biological expertise needed to construct and propagate them. Recent optimization efforts have resulted in simple 24-hour workflows for cell extract preparation47,48 that can be performed in parallel with energy mix preparation49,50. However, traditional cloning adds multiple days to the CFE prototyping timeline (Table 1)23. Quickly amplified PCR products from the commercial gene fragment can be used directly51, but this limits the number of prototyping experiments as only 1 &#181;g of DNA is produced, which corresponds to approximately five reactions (traditional 15 &#181;L volumes). With these additional steps of circularization and isothermal amplification, greater than milligram quantities of the DNA is possible (~5,000 reactions for 1 mg). This dramatically increases the number of tests that can be made in high-throughput screening of proteins or combinatorial enzyme networks (cell-free metabolic engineering); it also allows for effective preservation of the linear template library as high concentration DNA. Furthermore, an increased amount of template would be necessary to prototype larger quantities of protein needed for material science applications (protein-based fibers and hydrogels). Some limitations of linear templates can be overcome by using an extract from BL21 DE3 Star or using recently discovered methods to protect linear templates from degradation52,53,54. However, this does not address having limited stocks of vendor-produced DNA for PCR amplification or the issue of biological expertise and equipment needed for cloning.
This work presents a protocol explicitly designed to increase the amount of expression template that can be obtained from small quantities of vendor-produced gene fragments (typically 500-1000 ng of lyophilized powder). The described method does not require the skills necessary to perform traditional cloning in plasmids or transforming and propagating in living cells. Upon receiving a gene fragment in the mail, a user can produce enough templates for many cell-free reactions by employing isothermal rolling circle amplification (RCA) (Figure 1)23. While the amount of DNA received from the vendor may be enough for limited screening efforts, it is quickly depleted, and re-purchasing gene fragments is time consuming and costly. The method is also especially well-suited for genes that are toxic and difficult to clone in E. coli.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alaline</td>
			<td>Formedium</td>
			<td>DOC0102</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium glutamate</td>
			<td>MP Biomedicals</td>
			<td>MP21805951</td>
			<td></td>
		</tr>
		<tr>
			<td>Arginine</td>
			<td>Formedium</td>
			<td>DOC0106</td>
			<td></td>
		</tr>
		<tr>
			<td>Asparagine</td>
			<td>Formedium</td>
			<td>DOC0114</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspartic Acid</td>
			<td>Formedium</td>
			<td>DOC0118</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Axygen Sealing Film</td>
			<td>Corning</td>
			<td>PCR-SP</td>
			<td></td>
		</tr>
		<tr>
			<td>CMP</td>
			<td>Sigma</td>
			<td>C1006</td>
			<td></td>
		</tr>
		<tr>
			<td>Coenzyme A</td>
			<td>Sigma</td>
			<td>C3144</td>
			<td></td>
		</tr>
		<tr>
			<td>CutSmart Buffer</td>
			<td>NEB</td>
			<td>B7204S</td>
			<td>Provided with HindIII</td>
		</tr>
		<tr>
			<td>Cysteine</td>
			<td>Formedium</td>
			<td>DOC0122</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Clean and Concentrator Kit</td>
			<td>Zymo Research</td>
			<td>D4004</td>
			<td>Used for purifying DNA</td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>NEB</td>
			<td>N0447</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli tRNA</td>
			<td>Sigma (Roche)</td>
			<td>10109541001</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic Acid</td>
			<td>Sigma</td>
			<td>47612</td>
			<td></td>
		</tr>
		<tr>
			<td>Gene Fragment</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamic Acid</td>
			<td>Formedium</td>
			<td>DOC0134</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Formedium</td>
			<td>DOC0130</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Formedium</td>
			<td>DOC0138</td>
			<td></td>
		</tr>
		<tr>
			<td>GMP</td>
			<td>Sigma</td>
			<td>G8377</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>HindIII-HF</td>
			<td>NEB</td>
			<td>R3104L</td>
			<td></td>
		</tr>
		<tr>
			<td>Histidine</td>
			<td>Formedium</td>
			<td>DOC0142</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoleucine</td>
			<td>Formedium</td>
			<td>DOC0150</td>
			<td></td>
		</tr>
		<tr>
			<td>Leucine</td>
			<td>Formedium</td>
			<td>DOC0154</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysine</td>
			<td>Formedium</td>
			<td>DOC0158</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium glutamate</td>
			<td>Sigma</td>
			<td>49605</td>
			<td></td>
		</tr>
		<tr>
			<td>Methionine</td>
			<td>Formedium</td>
			<td>DOC0166</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtiter Plate (384 well)</td>
			<td>Greiner</td>
			<td>781906</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtiter Plate (96 well)</td>
			<td>Greiner</td>
			<td>655809</td>
			<td></td>
		</tr>
		<tr>
			<td>Multimode Plate Reader</td>
			<td>BioTek</td>
			<td>Synergy Neo2</td>
			<td></td>
		</tr>
		<tr>
			<td>NAD</td>
			<td>Sigma</td>
			<td>N8535</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoPhotometer</td>
			<td>Implen</td>
			<td>NP80</td>
			<td></td>
		</tr>
		<tr>
			<td>OneTaq DNA Polymerase</td>
			<td>NEB</td>
			<td>M0480</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Tube</td>
			<td>VWR</td>
			<td>20170-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylalanine</td>
			<td>Formedium</td>
			<td>DOC0170</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoenolpyruvate</td>
			<td>Sigma (Roche)</td>
			<td>10108294</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium glutamate</td>
			<td>Sigma</td>
			<td>G1501</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium oxalate</td>
			<td>Fisher Scientific</td>
			<td>P273</td>
			<td></td>
		</tr>
		<tr>
			<td>Proline</td>
			<td>Formedium</td>
			<td>DOC0174</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Serine</td>
			<td>Formedium</td>
			<td>DOC0178</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma</td>
			<td>S0266</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase Reaction Buffer</td>
			<td>NEB</td>
			<td>B0202S</td>
			<td>Provided with T4 DNA Ligase</td>
		</tr>
		<tr>
			<td>TempliPhi Amplification Kit</td>
			<td>Cytiva</td>
			<td>25640010</td>
			<td>Used for RCA</td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>Biorad</td>
			<td>C1000 Touch</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermoblock</td>
			<td>Eppendorf</td>
			<td>ThermoMixer FP</td>
			<td></td>
		</tr>
		<tr>
			<td>Threonine</td>
			<td>Formedium</td>
			<td>DOC0182</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptophan</td>
			<td>Formedium</td>
			<td>DOC0186</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrosine</td>
			<td>Formedium</td>
			<td>DOC0190</td>
			<td></td>
		</tr>
		<tr>
			<td>UMP</td>
			<td>Sigma</td>
			<td>U6375</td>
			<td></td>
		</tr>
		<tr>
			<td>Valine</td>
			<td>Formedium</td>
			<td>DOC0194</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid, enzymatic methods for amplification of minimal, linear templates for protein prototyping using cell-free systems

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

Expression of sfGFP from RCA templates was comparable to that of the pJL1 plasmid when using only 0.30 &#181;L of unpurified RCA DNA in a 15 &#181;L reaction (Figure 2A). In fact, doubling and tripling the amount of template appears to offer no benefit in BL21 DE3 Star extract, suggesting already saturated levels of the template at 0.30 &#181;L per reaction. Conversely, there appears to be a benefit to increasing the amount of RCA template when added to cell extract sourced from the SHuffle ...

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

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

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

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JoVE Journal

Bioengineering

2.6K Views.  Iowa State University. The study describes a protocol for creating large (µg-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

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

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

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

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 &#956;L batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of &#62; 260 &#956;g/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1&#8722;2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.

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

Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

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

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

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

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

Rapid Characterization of Genetic Parts with Cell-Free Systems

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

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