Name: Video: Rapid Characterization of Genetic Parts with Cell-Free Systems
Uploaded: 2021-08-30T14:00:00.000+00:00
Duration: 5 min

This work was made possible by the Office of the Secretary of Defense&#39;s Applied Research for the Advancement of Science and Technology Priorities program. We thank Scott Walper (Naval Research Laboratory) for providing the stock of sfGFP used, and Zachary Sun and Abel Chiao (Tierra Biosciences) for fruitful discussions related to prototyping with cell-free systems and related troubleshooting of acoustic liquid handling.

1. Preparation of cell extract
<ol>
	<li>Preparation of media
	<ol>
		<li>For 2xYT media: Add 16 g of tryptone, 10 g of yeast extract, and 5 g of NaCl to 900 mL of deionized water and adjust the pH to 7.0 with 5 M NaOH. Raise the solution volume to 1 L using deionized water and autoclave or filter sterilize. Alternatively, purchase 2xYT media.</li>
		<li>For S30B buffer: Prepare a solution of 14 mM Mg-glutamate, 60 mM K-glutamate, and 5 mM Tris in 2 L of deionized water. Use 2 M Tris to adjust the pH to 8.2, autoclave,&#160;and store at 4 &#176;C. Complete the solution by adding dithiothreitol (DTT) to a final concentration of 1 mM just before use.</li>
	</ol>
	</li>
	<li>Preparation of cells
	<ol>
		<li>Streak Escherichia coli BL21(DE3) Rosetta2 cells or other cell line of choice (see Cole et al.44 for a recent comprehensive review) onto an LB (Lysogeny Broth) agar plate and incubate at 37 &#176;C for 10-14 h.</li>
		<li>Use a single E. coli colony to inoculate 3 mL of 2xYT medium in a 10 mL culture tube. Incubate this tube at 37 &#176;C with shaking at 250 rpm for 8 h.</li>
		<li>Use 50 &#181;L from the 3 mL culture to inoculate 50 mL of 2xYT medium in a 500 mL flask. Incubate this flask at 37 &#176;C with shaking at 250 rpm for 8 h.</li>
		<li>Use 7.5 mL from the 50 mL culture to inoculate each of four 4 L baffled flasks containing 0.75 L of 2xYT medium. Incubate these flasks at 37 &#176;C with shaking at 220 rpm until they have reached an optical density at 600 nm of 2 to 4, after approximately 3-4 h.</li>
		<li>Harvest the cells from each flask by transferring them to 1 L containers and centrifuging at 5,000 x g for 12 min at 4&#160;&#176;C. Discard the supernatant by decanting into a waste container.</li>
		<li>Wash each cell pellet with 150 mL of ice-cold S30B buffer by completely resuspending them using a pipette to disrupt the cell mass, and then collect the cells again by centrifugation at 5,000 x g for 12 min. Discard the supernatant.</li>
		<li>Wash each cell pellet again in 40 mL of ice-cold S30B buffer by completely resuspending them and disrupting the cell mass using a pipette. Transfer the cells to pre-weighed 50 mL conical tubes and collect the cells again by centrifugation at 2,000 x g for 8 min&#160;at 4&#160;&#176;C. Discard the supernatant by decanting.</li>
		<li>Weigh the wet cell pellets. Flash-freeze the cell pellets by placing the tubes directly into liquid nitrogen and store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Cell lysis
	<ol>
		<li>Thaw the cell pellets on ice.</li>
		<li>Resuspend each cell pellet in 1.4 mL of S30B buffer per 1 g of the cell pellet by vortexing.</li>
		<li>Lyse the cells by French pressure cell at 640 psi at 4 &#176;C. Collect the lysate in microcentrifuge tubes on ice and add 3 &#181;L of 1 M DTT per 1 mL of lysate immediately after lysis. 
		NOTE: It is best to tap the French press release valve with a small metal rod to maintain even pressure and avoid sudden drops in pressure.</li>
		<li>Clear the lysate by centrifugation at 30,000 x g for 30 min at 4 &#176;C and discard the pellet after pipetting the supernatant to a new ice-cold microcentrifuge tube, taking care not to disrupt the pellet.</li>
		<li>Centrifuge the supernatant a second time at 30,000 x g for 30 min at 4 &#176;C. Pipette the resulting supernatant into an ice-cold microcentrifuge tube. Discard the pellet.</li>
		<li>Incubate the supernatant in a 37 &#176;C water bath for 1 h.</li>
		<li>Clear the supernatant by centrifugation at 15,000 x g for 15 min at 4 &#176;C and transfer the resulting supernatant to an ice-cold microcentrifuge tube, taking care not to disrupt the pellet.</li>
		<li>Centrifuge the supernatant a second time at 15,000 x g for 15 min at 4 &#176;C and transfer the resulting supernatant to an ice-cold microcentrifuge tube, taking care not to disrupt any remaining pellet.</li>
		<li>Distribute the supernatant in 100 &#181;L aliquots into 1.5 mL microcentrifuge tubes and flash freeze them by placing directly into liquid nitrogen. Store the supernatant at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Linear template preparation
<ol>
	<li>Primer design
	<ol>
		<li>Choose a core sequence as the PCR template. Include at a minimum a reporter sequence, such as sfGFP (superfolder Green Fluorescent Protein), LacZ, or Spinach aptamer. Include other parts that will be fixed across screened variants, such as terminators, promoters or RBSs, as appropriate for the design. 
		NOTE: Inclusion of a terminator is not always required for expression from linear DNA in cell-free systems.</li>
		<li>For the forward primers, choose a minimum of 20 bp matching the 5&#697; end of the core sequence as the 3&#697; end of the primer. If adding parts to the 5&#8242; end of the construct, design the remainder of the 5&#697; end of the primer to add the genetic parts of interest to the core sequence via PCR amplification (Figure 1A and Figure 2). 
		NOTE: Since primers above ~60 bp frequently increase dramatically in cost, multiple overlapping primers can be designed to add longer sequences or multiple parts. While multiple primers can be used in a single PCR reaction, performing multiple rounds of PCR is recommended.</li>
		<li>For the reverse primers, choose a minimum of 20 bp to match the 3&#8242; end of the core sequence as the 3&#697; end of the primer. If adding parts to the 3&#8242; end of the construct, design the remainder of the 5&#697; end of the primer to add the genetic parts of interest to the core sequence via PCR amplification (Figure 1A and Figure 2). Ensure that the reverse primer&#39;s annealing temperature is within 5 &#176;C of the annealing temperature of the entire forward primer.</li>
	</ol>
	</li>
	<li>Linear template amplification
	<ol>
		<li>Determine the number of PCR reactions to be performed based on the number of core sequences and calculate the amount of each component required using Table 1.</li>
		<li>Prepare the master mix according to Table 1 and store it on ice. Aliquot 30 or 40 &#181;L (see Table 1) of the master mix into the determined number of PCR tubes and add 10 &#181;L of each variable primer (i.e., primers encoding a part change, see Table 1) at 5 &#181;M to appropriately labeled PCR tubes.</li>
		<li>Place the PCR tubes into the thermocycler and run the following PCR program: 98 &#176;C for 3 min; 30 cycles of 98 &#176;C for 15 s, XX &#176;C for 20 s, 72 &#176;C for YY min;&#160;final extension at 72 &#176;C for 10 min. Then, hold the reaction at 4 &#176;C. 
		Where, XX represents the annealing temperature for the primer with the lower annealing temperature and YY represents the extension time calculated for the length of the amplicon based on the manufacturer&#39;s recommendations for the high-fidelity polymerase used. Optimize these conditions as needed for different primers and/or templates.</li>
		<li>(Optional) Add 1 &#181;L of DpnI restriction enzyme to digest the original template. Incubate the reaction at 37 &#176;C for 1 h. Perform this step only if the original template is plasmid DNA.</li>
		<li>Analyze 5 &#181;L of each PCR product by gel electrophoresis. Separate the product using a 1% agarose gel at 180 V for 20 min. Check for the correct band size, which will vary with the chosen core sequence and the length of the parts added.</li>
	</ol>
	</li>
	<li>Purify the linear template using a commercial PCR purification kit or by the preferred PCR cleanup method. If multiple bands were present by gel electrophoresis analysis, either optimize the PCR conditions or purify the correct molecular weight bands using a commercial gel extraction kit as per the manufacturer&#39;s recommendation.</li>
	<li>Quantify each DNA template using a spectrophotometer. Assess the DNA template quality by checking that the 260 nm/280 nm ratio is approximately 1.8.</li>
	<li>(Optional) Again separate a portion of the DNA template using a 1% agarose gel at 180 V for 20 min and ensure that any unwanted bands were removed during template purification.</li>
	<li>Use purified DNA templates immediately or store at -20 &#176;C.</li>
</ol>
3. Purified protein preparation
<ol>
	<li>Protein expression
	<ol>
		<li>For each protein to be expressed, assemble an appropriate expression construct. Codon-optimize the gene for expression in E. coli. Insert the gene into a pET-22b expression vector or other appropriate expression vector via the preferred plasmid assembly method. Transform the expression plasmid into BL21(DE3) Rosetta2 expression cells or other appropriate cell line.</li>
		<li>For each protein, use a single colony to inoculate 3 mL of LB medium in a 10 mL culture tube. Incubate these tubes at 37 &#176;C with shaking at 250 rpm overnight.</li>
		<li>Inoculate a 2 L flask containing 750 mL of LB medium with 1 mL of the overnight culture. Incubate these flasks at 37 &#176;C with shaking at 250 rpm until they reach an OD600 of 0.6-1.0.</li>
		<li>Induce protein expression by adding 0.75 mL of 1 M isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) in water to each flask and continue to incubate these flasks at 37 &#176;C with shaking at 250 rpm for 4 h.</li>
		<li>Harvest the cells from each flask, using a 1 L centrifuge bottle, by centrifugation at 5,000 x g for 12 min. Discard the supernatant.</li>
		<li>Transfer the pellets to a 50 mL conical tube and weigh each pellet. Flash-freeze the cells in liquid nitrogen and store them at -80 &#176;C or proceed to step 3.2. 
		NOTE: 2-5 g of cells per 0.75 mL are expected to result from this step.</li>
	</ol>
	</li>
	<li>Protein purification by nickel affinity column chromatography
	<ol>
		<li>Prepare the lysis buffer by combining 50 mM Tris-Cl, 500 mM NaCl, and 5 mM imidazole. Adjust to pH 8.0.</li>
		<li>Prepare the wash buffer by combining 50 mM Tris-Cl, 500 mM NaCl, and 25 mM imidazole. Adjust to pH 8.0.</li>
		<li>Prepare the elution buffer by combining 50 mM Tris-Cl, 500 mM NaCl, and 250 mM imidazole. Adjust to pH 8.0.</li>
		<li>Prepare the dialysis buffer by combining 50 mM NaHPO4, 100 mM NaCl, and 2% DMSO. Adjust to pH 7.5.</li>
		<li>Thaw the cell pellets by placing the tubes in room temperature water. Add 5 mL of the lysis buffer per 1 g of the cell pellet and resuspend by vortexing.</li>
		<li>Lyse the cells by sonication. Separate the cell homogenate so that there is no more than 30 mL per 50 mL conical tube and keep each tube on ice. Lyse the cells using a sonicator with a 0.16 cm diameter probe in 15 s rounds with 30 s breaks, 10 times. 
		NOTE: Avoid foaming, as this denatures the protein. The formation of foam can be avoided by keeping the sonicator tip at least 2/3 submerged in the lysate while it is operational. Other methods of cell lysis besides sonication are also possible44.</li>
		<li>Clear the lysate by centrifugation at 15,000 x g for 30 min at 4 &#176;C and place the supernatant into a new 50 mL conical tube.</li>
		<li>Add 1 mL of nickel-nitrilotriacetic acid (Ni-NTA) resin for each 5 mL of supernatant. Divide the cell lysate/Ni-NTA slurry so that there is no more than 36 mL per 50 mL conical tube. Incubate at 4 &#176;C on a tube rotator at 10 rpm for 1 h.</li>
		<li>Load the resin by decanting the cell lysate/Ni-NTA slurry into a 2 mL bed volume chromatography column and collect the eluant if needed for further analysis, otherwise, discard. Wash the resin with 10 resin bed volumes of wash buffer.</li>
		<li>Collect the protein by adding three resin bed volumes of elution buffer to the column and concentrate the volume to 1.5 mL using a centrifugal concentrator with the appropriate molecular weight cut-off membrane for each protein.</li>
		<li>Dialyze the protein against 2 L of dialysis buffer at 4 &#176;C for 1 h. Dialyze the protein again against 2 L of dialysis buffer overnight at 4 &#176;C.</li>
		<li>Quantify the protein using its molar extinction coefficient and absorbance at 280 nm. Analyze the protein for purity by separating it using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Store the protein at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Cell-free protein synthesis
<ol>
	<li>Preparation of CFPS reaction mixture
	<ol>
		<li>Prepare the Supplement Mix by following the Amino Acid Solution Preparation, Energy Solution Preparation, and Buffer Preparation steps in Sun et al.42. Store separately or combined at -80 &#176;C in aliquots. Ensure that final concentrations match those described in Sun et al.42 in the Experimental Execution of a TX-TL Reaction section.</li>
		<li>Prepare an additive to protect linear DNA from degradation. If using GamS33,45, prepare via steps in section 3 above, or obtain from a commercial vendor; for other approaches, check the corresponding literature46,47,48. Alternatively, use a CFPS system that does not require additives49.</li>
		<li>Prepare T7 polymerase, repressor proteins, and other additives using the steps in section 3 above or obtain from a commercial vendor.</li>
		<li>Determine the number of CFPS reactions to be performed and calculate the amount of each component required using Table 2. Modify the concentrations of the components, including adding or removing components as needed, and adjust the amount of water such that the final volume of each reaction mixture is always 10 &#181;L. Similarly, modify the master mix to facilitate dispensing of other components by acoustic liquid handling as desired (see the Discussion section).</li>
		<li>Thaw all the components on ice and prepare a master mix by mixing each component as calculated above. Mix all the components thoroughly by pipette. Pay careful attention to avoid precipitation, especially for the amino acid mixture. Keep the master mix on ice.</li>
		<li>Chill a 384-well plate on ice and distribute the master mix in 9 &#181;L aliquots into each well. 
		NOTE: It is possible to distribute these components by acoustic liquid handling, though care should be taken to ensure proper dispensing (see the Discussion section for troubleshooting).</li>
	</ol>
	</li>
	<li>Distribution of additional components by acoustic liquid handling
	<ol>
		<li>Calculate the amount of repressor protein (and other optional components) required for all the CFPS reactions.</li>
		<li>Thaw the repressor protein on ice and distribute it into an acoustic liquid handling source plate or other appropriate plate. Ensure that the appropriate amount of dead volume required for the type of source plate used is included.</li>
		<li>Distribute the repressor protein in 1 &#181;L volumes into the appropriate wells via the liquid handler. For more information on distribution troubleshooting, see the Discussion section.</li>
	</ol>
	</li>
	<li>Standard curves
	<ol>
		<li>Include a serial dilution of the purified reporter (see section 3 for protein purification)41 or appropriate chemical standard50&#160;on the plate to enable comparison of results with other studies and other labs. Choose a range of concentrations appropriate for the reporter used and expected expression range of the experiments.</li>
	</ol>
	</li>
	<li>Running CFPS reactions
	<ol>
		<li>Pre-warm the plate reader to 30 &#176;C. Set the plate reader to read at settings appropriate for the reporter used in the core sequence without&#160;shaking steps. 
		NOTE: While 30 &#176;C is used here, 29 &#176;C and 37 &#176;C are also commonly used and work well with this protocol. Other temperatures may be preferred for alternative cell-free reaction preparations. For read intervals, 10 min is sufficient to achieve a good resolution for the representative data presented here; however, other resolutions may be better depending on the reporter protein and the particular CFPS recipe.</li>
		<li>(Optional) Run a test reaction first to set the appropriate gain or sensitivity setting to capture the change in fluorescence without signal overflow.</li>
		<li>Seal the 384-well plate with an impermeable plastic sealable lid to prevent evaporation. If possible, on the instrument, set a 1 &#176;C vertical temperature gradient to limit condensation on the seal. Place the 384-well plate on the plate holder and begin reading.</li>
	</ol>
	</li>
</ol>

RMM has a financial stake in Tierra Biosciences, a private company that makes use of cell-free technologies such as those described in this article for protein expression and screening.
The other authors have nothing to disclose.

The protocols described here provide a cost-effective and rapid means to screen genetic parts via the expression of a reporter protein by CFPS. Well-characterized genetic parts are crucial to the design of predictable genetic circuits with useful function. This methodology increases throughput and decreases the time needed to screen new genetic parts by removing the requirement to work in living cells, while retaining functionality that mirrors the cellular environment by retaining the metabolic process of protein expression in the cell lysate. Our protocol can be performed in 1 day after receipt of primers (~2.5-6 h for reaction preparation, 2-16 h for CFPS reaction; Figure 1), compared to at least 3 days for traditional cloning (1 day each for construct assembly and transformation, sequence verification of clones, and culturing of cells for assessment). We further estimate that the cost per construct using linear DNA is roughly one-third of the traditional cloning ($78 vs. $237; Supplementary Table 1) methods. Commercial synthesis services currently quote a minimum of 4 business days depending on size, though they would have similar costs to our method if linear fragments are screened directly in CFPS ($78 vs. $91); we have not verified this approach. The cost to evaluate a part with CFPS is small compared to the generation of the template DNA ($0.05/reaction22 vs. $78 per template), though it should be noted that the startup costs for bulk reagents and lysis equipment is at least several thousand dollars. The use of an acoustic liquid handler only marginally improves costs by enabling smaller volumes down to 0.5 &#181;L40; the more significant advantage is the reduction of time to prepare reactions (~10 min vs. up to 1 h, depending on the number of reactions), especially when preparing a large number of reactions raises concerns of prepared reaction sitting for extended times before incubation.
While rapid and cost-effective, the limitations on when CFPS prototyping adequately predicts in vivo function remain to be seen. For example, any cross-reactivity with genomic DNA will not be detected due to removal of the host genome during the production of the CFPS system. Also, component concentrations can be 1-2 orders of magnitude lower in CFPS than in cells51, which is likely to affect the behavior of some parts as a result of different macromolecular crowding conditions. Further, the ability of linear DNA to predict in vivo function may be limited, for example, when DNA secondary structure plays an important role. A final limitation is that constructs are not sequence-verified before testing for functions. There may be cases where the part characterized is not actually aligned with the intended theoretical sequence. All of these limitations can be mitigated by validating a subset of the parts screened by this method in the intended in vivo application.
We originally developed this methodology to investigate the effects of changing the operator position on hybrid T7-tetO promoters32. We have presented the protocols here in a more generic format, such that they can be applied to promoters, operators, ribosome binding sequences, insulators, and terminators. These genetic parts can be added to the 5&#697; or 3&#697; end of the reporter gene by PCR using primers for each design, obviating the need for synthesis or cloning of each variant to test. The resulting PCR products serve as template DNA for evaluation via the expression of a reporter protein. In our work, the affinity purification protocol provided here was used for TetR and GamS. The same procedure can be used for the expression and purification of other repressors, activators, polymerases, sigma factors, and other proteins cognate to a genetic part of interest, although modifications may be needed for the desired protein being expressed. Purification and titration of these proteins into CFPS reactions enables a more detailed characterization of a particular genetic part. Finally, numerous alternative CFPS protocols exist and each should be amenable to the parts screening portion of methodology. As an example, we do not include a dialysis step in this protocol, which others have found to be important for expression from native bacterial promoters22. Varying the concentrations of underlying constituent components of the CFPS is also possible. The use of liquid handling enhances the ability to test the myriad conditions by increasing throughput and decreasing the materials required34,35.
One area that can require significant troubleshooting is optimization of the acoustic liquid handler. Acoustic liquid handler dispensing should be optimized for each component being transferred and it is strongly recommended to run controls to verify proper distribution and reproducibility before collecting data. The ideal source plate type and liquid class setting will depend on the specific liquid to be dispensed and its components. It is not recommended to use amine-coated plates to dispense DNA, as the amine coating may interact with the DNA. It should also be noted that the ability to dispense higher concentrations of certain components may depend on the acoustic liquid handler model. A test liquid transfer may be conducted by dispensing onto a foil plate seal to visualize successful droplet formation; however, this test provides limited information and droplets from different settings may appear identical. The use of a water-soluble dye, such as tartrazine, may be used to more accurately verify the correct volume is dispensed with a given setting or workflow (see Representative Results). Optimal programming of liquid transfers can also influence the accuracy and consistency of data generated; for transfers &#62;1 &#181;L from one source well to one destination well, we have found that sequential transfers of &#8804;1 &#181;L should be programmed to reduce systematic well-to-well variability (Figure 4). Lastly, theoretical and actual source well dead volumes can vary dramatically depending on the source plate type, liquid class setting, and components of the specific liquid; using the acoustic liquid handler survey function to assess the well volumes prior to running a program may help gauge how accurately the instrument is able to measure a particular liquid.
CFPS reaction performance can vary when comparing results between different users, batches of materials, plate readers, and laboratories41. For instances where such comparisons are required while prototyping genetic circuits, we recommend including internal control reactions with standard constitutive promoters in each reaction plate to help normalize results across experimental setups. The method of DNA preparation can also contribute majorly to CFPS activity; the inclusion of an ethanol precipitation step is recommended. In addition, the optimal reaction composition can vary by the batch of extract34. Optimal magnesium glutamate and potassium glutamate concentrations, in particular, have been shown to vary by batch42 or with the promoter or reporter protein used24. Concentrations of these components should be optimized by screening across several concentrations of each component per genetic construct and per cell extract preparation to determine the optimal conditions for protein expression. Finally, best practices for consistent CFPS reaction performance include thorough mixing, careful pipetting, and consistency in the preparation of each reagent component.
Beyond characterization of individual parts, the same method can be used to screen combinations of parts that form complex circuits, such as logic circuits16 or oscillators52,53. This method can also be applied to screening and optimizing biosensors for applications in epidemiological diagnostics54,55,56,57 or hazard detection and quantification3,58,59. The application of AI-driven techniques such as active learning34 can also be paired with the high-throughput nature of this method to drive rapid exploration of complex biological design spaces. Ultimately, we envision this approach supporting accelerated development times for new genetic designs in synthetic biology.

A core effort of synthetic biology is to develop genetic tool kits containing well-characterized parts, which can be used to construct genetic circuits1 that carry out useful functions when deployed in microbes or cell-free lysates. Areas in which such genetic circuits have gained traction are sensing2,3,4, human performance5,6, biofuels7,8, materials production9,10, and cellular computing11. Registries of standardized genetic parts have been established12 to catalog new and existing parts into categories such as promoters, operators, coding sequences, and terminators, to name just a few. Efforts such as the iGEM (international Genetically Engineered Machines) competition13 have been instrumental in characterizing and cataloging these genetic parts. Many methods have been developed to facilitate the rapid assembly of these parts into useful genetic circuits14,15. Software has even been developed to automate the composition of well-characterized parts into circuits that achieve a desired function16. However, the assembly of useful genetic circuits with predictable functions rests on the presumption that the genetic tool kits contain well-characterized genetic parts. Due to the necessity of these tool kits toward the advancement of synthetic biology, numerous efforts to better catalog circuits and parts with appropriate characterization data have been described17,18,19,20,21.
One approach to characterizing genetic components makes use of cell-free protein synthesis (CFPS) systems, which reconstitute cellular functions such as transcription and translation ex vivo22. Several studies have demonstrated the potential of CFPS for prototyping genetic components23,24,25,26,27,28,29,30,31,32 whether for direct applications in cell-free systems or to predict the function of genetic constructs in cells, such as the relative activity of parts within a library29, metabolic pathway optimization27, and cellular burden30. Advantages to prototyping in CFPS versus cells highlighted by these studies include avoiding time-consuming cloning, precise control over the concentration of DNA and other reaction components, and the ability to easily mix and match multiple DNA constructs. The advantage of avoiding cloning is especially apparent when using linear DNA templates, which enables new constructs to be assembled by in vitro methods that take hours instead of days33. The ability to manipulate the concentration of DNA constructs and other components simply by pipetting makes the approach even more attractive by enabling high-throughput experimentation powered by liquid handling robots34,35. While successes using CFPS for prototyping have been reported, it is important to note that it remains to be seen under what contexts CFPS results can reliably predict functionality in cells.
Here, we present a method for CFPS prototyping that emphasizes the advantages in speed, throughput, and cost compared to traditional cell-based approaches. The approach is derived from our previous work where we used CFPS to rapidly characterize a library of T7 promoter variants regulated by the transcription factor TetR32, significantly expanding on the small handful of regulated T7 promoter variants that were available in the literature at the time36,37. Others have, since then, further expanded the range of such promoters38. In our method, genetic construct assembly is accelerated by using PCR to amplify template DNA via primers that add variant genetic parts to a reporter gene. Acoustic liquid handling in 384-well plates is used to increase throughput and decrease the volume of materials required. Previous work has demonstrated successful use of acoustic liquid handling at significantly lower volumes39,40 with variability comparable to manual pipetting of larger volumes41. In addition to the method, we provide troubleshooting information and an assessment of potential cost and time savings. Note that while we include a protocol for producing cell-free lysates based on Sun et al.42 here, numerous other commercial kits and protocols43,44 should also work. Similarly, while we demonstrate the method for the characterization of promoter variants32, other parts can be interchanged by PCR amplification, such as riboregulators, Ribosome Binding Sites (RBSs), insulators, protein tags, and terminators. We hope that this methodology can help the synthetic biology community continue to grow the number of characterized parts for the assembly of predictable genetic circuits with useful function.

<table><tbody><tr><td>2x YT medium</td><td>Sigma-Aldrich</td><td>Y2377-250G</td><td>Alternative to making 2xYT media</td></tr><tr><td>Agar</td><td>Bacto</td><td>214010</td><td>For plating cells</td></tr><tr><td>Chromatography column (5 cm diameter)</td><td>BIO-RAD</td><td>731-1550</td><td>Used for protein purification.</td></tr><tr><td>Destination plate</td><td>Thermo Scientific Nunc plate</td><td>142761</td><td>For CFPS reactions</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2650</td><td>For dialysis buffer</td></tr><tr><td>DpnI</td><td>NEB</td><td>R0176L</td><td>For digestion of plasmid templates</td></tr><tr><td>DTT</td><td>Roche</td><td>20871723</td><td>S30 Buffer B</td></tr><tr><td>E. coli BL21(DE3) Rosetta2</td><td>Novagen</td><td>70954</td><td>Cell line used for production of lysate and purified proteins</td></tr><tr><td>Echo acoustic liquid handler</td><td>Labcyte</td><td>525</td><td>Acoustic liquid handler</td></tr><tr><td>French pressure cell</td><td>Thermo Spectronic</td><td>FA-078</td><td>For lysing cells for CFPS</td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>56750</td><td>For buffers</td></tr><tr><td>Impermeable plastic sealable lid</td><td>Thermo</td><td>232702</td><td>Plate seal</td></tr><tr><td>IPTG</td><td>RPI</td><td>I56000-25.0</td><td>Used for protein induction.</td></tr><tr><td>K-Glu</td><td>Sigma-Aldrich</td><td>g1501-500G</td><td>S30 Buffer B</td></tr><tr><td>Labcyte Echo source plate</td><td>Labcyte</td><td>PL-05525</td><td>For use with Echo acoustic liquid handler</td></tr><tr><td>Mg-Glu</td><td>Sigma-Aldrich</td><td>49605-250G</td><td>S30 Buffer B</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653-250G</td><td>For buffers</td></tr><tr><td>NaHPO4</td><td>Sigma-Aldrich</td><td>71505</td><td>For dialysis buffer</td></tr><tr><td>NaOH</td><td>Mallinckrodt Chemicals</td><td>7708-10</td><td>For making 2xYT media. Currently not produced by Mallinckrodt. Alternate: Sigma-Aldrich S0899</td></tr><tr><td>Ni-NTA resin</td><td>Invitrogen</td><td>R901-15</td><td>For production of purified proteins</td></tr><tr><td>PCR H2O</td><td>Ambion</td><td>AM9937</td><td>PCR of linear templates</td></tr><tr><td>Plate Reader</td><td>BioTek</td><td>H10</td><td>Plate reader used</td></tr><tr><td>Q5 PCR Master Mix</td><td>NEB</td><td>M0494S</td><td>PCR of linear templates</td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28606</td><td>PCR of linear templates</td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28004</td><td>PCR of linear templates</td></tr><tr><td>QSonica Ultrasonic Processor</td><td>Qsonica</td><td>Q700</td><td>Cell disruption during protein purification</td></tr><tr><td>RTS Amino Acid Sampler</td><td>biotechrabbit</td><td>BR1401801</td><td>Updated supplier from Sun et al.</td></tr><tr><td>Tris</td><td>MP</td><td>819623</td><td>S30 Buffer B</td></tr><tr><td>Tris-Cl</td><td>Sigma-Aldrich</td><td>T5941</td><td>For buffers</td></tr><tr><td>Tryptone</td><td>Fluka</td><td>T7293</td><td>For making 2xYT media</td></tr><tr><td>Yeast Extract</td><td>Bacto</td><td>212750</td><td>For making 2xYT media</td></tr></tbody></table>

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.

To demonstrate the utility of our methods, we present results that describe the effects of proximity of the tetO sequence to the T7 promoter on the regulation of T7 RNAP-driven expression. The full results and their implications can be found in the work of McManus et al.32. The workflow is described in Figure 1. Fifteen linear templates, varying only in the distance of the T7 promoter relative to the tetO sequence, were prepared by PCR-amplifying the sfGFP reporter with primers designed to add each promoter variant (Figure 2) as described in section 2 of the protocol. CFPS reaction components and reactions were prepared following the protocol. The expression of sfGFP was measured from each template with a titration of 12 different concentrations of the TetR protein, in triplicate, using an acoustic liquid handler. At 36 CFPS reactions per template and 15 templates, a total of 540 reactions for the entire set of T7-tetO combinations were performed. The entire evaluation was carried out on two plates in two plate readers. Analysis of this data showed that the T7 RNAP downregulates T7-driven expression equally up through 13 bp downstream from the start of the T7 transcript (Figure 3). This result has implications for the future design of regulatable T7-driven gene circuits by describing a putative window for an effective repression of T7 by other repressors. Comparison of results from the protocol described here with DNA prepared by traditional cloning revealed a small but statistically significant difference in the degree of TetR repression between formats. We hypothesized that non-specific binding of TetR to the vector DNA could explain the observed difference. Experimental results showed that addition of linear vector DNA to reactions with linear template DNA reduced the difference to non-statistical significance, though it did not rule out contributions from other factors, such as differences in periodicity of the DNA helix for linear vs. circular formats, which, in turn, could affect TetR binding. Depending upon the application, the use of linear template may require additional validation.
We further include representative data on potential issues with accurate dispensing using acoustic liquid handling (Figure 4). A solution of 1x phosphate buffered saline (PBS), pH 7.4 containing 0.25 mM tartrazine dye was used to evaluate two methods of programming an acoustic liquid handler to dispense volumes &#62;1 &#181;L. Following liquid dispensing, the destination plate was sealed and centrifuged at 1,500 x g for 1 min, and the absorbance at 425 nm measured with a plate reader. Representative results of nine experiments are shown and demonstrate more consistent dispensing across the series of eight destination wells when the 5 &#181;L transfer is divided into separate 1 &#181;L dispenses. Based on these observations, it is recommended that transfers &#62;1 &#181;L be broken down into multiple transfers of &#8804;1 &#181;L.&#160;See the Discussion section for more details on troubleshooting this important aspect of the protocol.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62816/62816fig01.jpg" /> 
Figure 1: Single-day workflow for the evaluation of promoter parts in cell-free extract. (A) A reporter is PCR-amplified using primers containing genetic parts to be evaluated (2-5 h). (B) The cell-free reaction mix is prepared as detailed in the protocol and distributed into a 384-well plate with the PCR-amplified templates (30 min). (C) Acoustic liquid handling is used to distribute additional components, which can include repressor proteins, effector molecules, and any other conditional effectors (10 min). (D) Reporter protein expression from each reaction is measured in a plate reader (2-16 h, depending on the CFPS recipe and construct). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62816/62816fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62816/62816fig02.jpg" /> 
Figure 2: Primer design for adding genetic parts to a reporter gene by PCR amplification. (A) The sfGFP reporter gene (green) will be amplified to add an RBS (red) and a T7 promoter (blue) by PCR. (B) The sfGFP (green) and an RBS (red) will be amplified to add a tetO sequence (gold) and a T7 promoter (blue) by PCR. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62816/62816fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62816/62816fig03.jpg" /> 
Figure 3: The effect of tetO position on the regulation of a T7-driven expression. Normalized maximum repression values for linear and circular template as a function of tetO position. Traces represent the mean and standard deviations for three replicates. This figure has been modified from McManus et al.32 under a Creative Commons CC-BY license. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62816/62816fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62816/62816fig04.jpg" /> 
Figure 4: Using tartrazine dye to validate liquid dispensing with an acoustic liquid handler. Black bars indicate dispensing 5 &#181;L of tartrazine solution from a single source well into each of the eight consecutive destination wells of a 384-well plate using a single programming command. Gray bars indicate dispensing 1 &#181;L from a single source well into each of eight consecutive destination wells using a single programming command, and then repeating this step four times for a total of 5 &#181;L dispensed in each destination well. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62816/62816fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component Name</td>
			<td>Volume for 1 reaction (&#181;L)</td>
			<td>Volume for 110% of X number of reactions (&#181;L)</td>
		</tr>
		<tr>
			<td>Q5 PCR Premix</td>
			<td>25</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>4</td>
			<td></td>
		</tr>
		<tr>
			<td>Template (1&#8211;3 ng/&#181;L)</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>(if fixed1) Forward Primer (5 &#181;M)</td>
			<td>0 or 10</td>
			<td></td>
		</tr>
		<tr>
			<td>(if fixed1) Reverse Primer (5 &#181;M)</td>
			<td>0 or 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Master Mix Total:</td>
			<td>30 or 40</td>
			<td></td>
		</tr>
		<tr>
			<td>(if variable1) Forward Primer (5 &#181;M)</td>
			<td>0 or 10</td>
			<td></td>
		</tr>
		<tr>
			<td>(if variable1) Reverse Primer (5 &#181;M)</td>
			<td>0 or 10</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Worksheet for the preparation of reagents for PCR reactions. Values in the rightmost column can be filled in by users depending on the intended number of reactions. 1Variable primers contain a specific part to be added in the PCR reaction and can be the forward primer, reverse primer, or both. Fixed primers do not add a part and can be the forward primer or reverse primer but not both.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component Name</td>
			<td>Volume for 1 reaction (&#181;L)</td>
			<td>Volume for 110% of X number of reactions (&#181;L)</td>
		</tr>
		<tr>
			<td>Cell Extract</td>
			<td>4.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Supplement Mix</td>
			<td>3.3</td>
			<td></td>
		</tr>
		<tr>
			<td>GamS Protein (207 &#181;M)</td>
			<td>0.15</td>
			<td></td>
		</tr>
		<tr>
			<td>Template DNA (20 nM)</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 Polymerase (13 mg/mL)</td>
			<td>0.12</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>0.73 (this number may vary)</td>
			<td></td>
		</tr>
		<tr>
			<td>Master Mix Total:</td>
			<td>9</td>
			<td></td>
		</tr>
		<tr>
			<td>Repressor Protein:</td>
			<td>1</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Worksheet for the preparation of reagents for CFPS reactions. Values in the rightmost column can be filled in by users depending on the intended number of reactions.
Supplementary Table. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62816/Table S1 - cost estimates_REV EDITS.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Rapid Characterization of Genetic Parts with Cell-Free Systems at JoVE.com

Rapid Characterization of Genetic Parts with Cell-Free Systems

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.

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the ...

rapid-characterization-of-genetic-parts-with-cell-free-systems

Nanyang Technological University

Research

JoVE Journal

Bioengineering

1.8K Views.  US Army Combat Capabilities Development Command Army Research Laboratory. Onze methode maakt de snelle karakterisering van genetische delen mogelijk met behulp van celvrije expressiesystemen en PCR in plaats van klonen. Het belangrijkste voordeel van deze techniek is dat wat meestal dagen tot weken in cellen duurt, met behulp van deze methode in uren tot dagen kan worden gedaan. Ons protocol bevat stappen om uw eigen celvrije expressiesystemen te maken, wat op een aantal plaatsen lastig kan zijn en aanpassingen kan vereisen, afhankelijk van de use case.Wanne...

Video: Rapid Characterization of Genetic Parts with Cell-Free Systems

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Genetics

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Biochemistry

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., ...

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Chemistry

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

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 ...

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.

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 ...

Rapid Development of Cell State Identification Circuits with Poly-Transfection

Mammalian genetic circuits have demonstrated the potential to sense and treat a wide range of disease states, but optimization of the levels of circuit components remains challenging and labor-intensive. To accelerate this process, our lab developed poly-transfection, a high-throughput extension of traditional mammalian transfection. In poly-transfection, each cell in the transfected population essentially performs a different experiment, testing the behavior of the circuit at different DNA copy numbers and allowing users to analyze a large number of stoichiometries in a single-pot reaction. So far, poly-transfections that optimize ratios of three-component circuits in a single well of cells have been demonstrated; in principle, the same method can be used for the development of even larger circuits. Poly-transfection results can be easily applied to find optimal ratios of DNA to co-transfect for transient circuits or to choose expression levels for circuit components for the generation of stable cell lines.
Here, we demonstrate the use of poly-transfection to optimize a three-component circuit. The protocol begins with experimental design principles and explains how poly-transfection builds upon&#160;traditional co-transfection methods. Next, poly-transfection of cells is carried out and followed by flow cytometry a few days later. Finally, the data is analyzed by examining slices of the single-cell flow cytometry data that correspond to subsets of cells with certain component ratios. In the lab, poly-transfection has been used to optimize cell classifiers, feedback and feedforward controllers, bistable motifs, and many more. This simple but powerful method speeds up design cycles for complex genetic circuits in mammalian cells.

Complex genetic circuits are time-consuming to design, test, and optimize. To facilitate this process, mammalian cells are transfected in a way that allows the testing of multiple stoichiometries of circuit components in a single well. This protocol outlines the steps for experimental planning, transfection, and data analysis.

Mammalian genetic circuits have demonstrated the potential to sense and treat a wide range of disease states, but optimization of the levels of circuit ...

Rapid Characterization of Genetic Parts with Cell-Free Systems

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