Name: synthesis of infectious bacteriophages in an e. coli-based cell-free expression system
Uploaded: 2017-08-17T15:00:00
Description: Watch this Scientific Journal Video about Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System at JoVE.com

This material is based on work supported by the Office of Naval Research award number N00014-13-1-0074 (to V.N.), the Human Frontier Science Program grant number RGP0037/2015 (to V.N.), and the Binational Science Foundation grant 2014400 (to V.N.).

NOTE: The following amplification steps and extraction method are largely generalizable for many double-stranded DNA phage, e.g., bacteriophage T7, enterobacteria phage T4, or enterobacteria phage &#955; (L). They are predominantly used for a phage whose genome is not readily available for purchase from commercial sources.
1. Phage Amplification
NOTE: The single-plaque, multi-cycle (SPMC) phage production technique is well described for the T4 phage in Chen, ...

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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+and+prototyping+genetic+networks+with+cell-free+transcription-translation+reactions.">Characterizing and prototyping genetic networks with cell-free transcription-translation reactions.</a> Methods. , (2015).</li><li>Shin, J., Jardine, P., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Replication,+Synthesis,+and+Assembly+of+the+Bacteriophage+T7+in+a+Single+Cell-Free+Reaction.">Genome Replication, Synthesis, and Assembly of the Bacteriophage T7 in a Single Cell-Free Reaction.</a> ACS Synthetic Biology. 1 (9), 408-413 (2012).</li><li>Garamella, J., Marshall, R., Rustad, M., Noireaux, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+All+E.+coli+TX-TL+Toolbox+2.0:+A+Platform+for+Cell-Free+Synthetic+Biology.">The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology.</a> ACS Synth Biol. , (2016).</li><li>Karzbrun, E., Shin, J., Bar-Ziv, R. 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Following the technique in Chen, et al.14 for SPMC, a critical step is reached when determining the proper conditions for superinfection. The parameter that most closely controls a host strain&#39;s ability to withstand superinfection is frequently the initial concentration of the infecting phage. The host cells must be in logarithmic growth phase before initial infection with a very small amount of phage. Eventually, the phage will also reach logarithmic growth and the goal is to allow t...

Over the past decade, the cell-free expression technology has been engineered to address novel applications in emergent multidisciplinary research areas related to synthetic and quantitative biology. Originally used to express proteins independent of a living organism, new cell-free TXTL systems have been developed for both basic and applied sciences1,2, broadening considerably the scope of this technology. The new generation of TXTL platforms has been designed to be user friendly, more efficient (reaching 2 mg/mL of protein synthesis in batch mode3), more versatile at the level of transcription4, and modular so as to easily integrate novel natural or synthetic functions that expand the capabilities of existing biological systems5,6. In particular, cell-free TXTL systems have become handy for the rapid prototyping of genetic programs, such as regulatory elements or small genetic circuits7,8,9, by reducing the design-build-test cycle to a few days. Remarkably, the new TXTL systems are also capable of processing large DNA programs such as the complete synthesis of coliphages10,11, demonstrating strong enough performances to support the reconstitution of active genomic DNA encoded living entities.
TXTL systems present many technical advantages compared to traditional in vitro constructive biochemical assays. Cell-free TXTL links the process of gene expression to the final product in a reduced and open environment, as opposed to the complex cytoplasm of a living cell. TXTL uses DNA to reconstruct biochemical systems in vitro, which, with modern DNA assembly techniques, is affordable and fast in addition to not requiring fastidious protein purification steps. Cell-free expression provides direct access to most of the components in the biochemical reactions, thus allowing a deeper dissection of the molecular interactions12. In a TXTL reaction, one can change the biochemical and biophysical settings at will, which is almost impossible in a living cell. Given these advantages and recent improvements, the TXTL technology is becoming more and more popular as an alternative platform for synthetic and quantitative biology. While the research community using TXTL is rapidly growing and TXTL is becoming a standard technology in bioengineering, it is essential to understand how to use such platforms so as to develop adequate practices related to the execution of TXTL reactions and to the interpretation of the results.
In this article, we describe how to use an all E. coli TXTL system to synthesize, in one-pot reactions, bacteriophages from their genome11, such as MS2 (RNA, 3.4 kb), &#934;&#935;174 (ssDNA, 5.4 kb), and T7 (dsDNA, 40 kb). We show how the amount of phages synthesized changes with respect to some of the biochemical settings of the reactions (magnesium and potassium concentrations). Molecular crowding, emulated through a range of PEG 8000 concentrations, has a dramatic effect on phage synthesis over several orders of magnitude. The realization of such large biochemical systems in single test tube reactions that recapitulate concurrently the processes of transcription, translation, and self-assembly, is interesting for addressing basic questions related to biology and biophysics10 (gene regulation, self-assembly), as well as for developing applications, such as repurposing phage functions to build new nanostructures13. In addition to a practical on TXTL, we provide methods for phage amplification, genome extraction and purification, and phage quantification by the plaque assay. The methods presented in this manuscript are appropriate for researchers who use E. coli extract based cell-free systems and are interested in bacteriophages.
The protocols presented in this work can be summarized as follow: 1) Phage amplification (Day 1: prepare inoculation cells, day 2: single plaque, multiple phage growth, and concentration, and day 3: purification of phage), 2) Double-stranded genome DNA extraction (phenol/chloroform extraction), and 3) Cell-free phage reaction and titer experiment (Day 1: plate host cells and make agar plates, day 2: cell-free reaction and host cell pre-culture, and day 3: host cell culture and phage titer).

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			<td class="xl21" height="21" style="width:162pt;height:15.75pt" width="216">Ultracentrifugation tubes</td>
			<td class="xl21" style="width:168pt" width="224">Beckman Coulter</td>
			<td class="xl21" style="width:219pt" width="292">344057</td>
			<td class="xl20" style="width:125pt" width="167"></td>
		</tr>
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			<td class="xl21" height="21" style="height:15.75pt">Conical tubes</td>
			<td class="xl22" style="width:168pt" width="224">Falcon</td>
			<td class="xl21">352070</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Gradient maker</td>
			<td class="xl21">BioComp Gradient Master</td>
			<td class="xl22" style="width:219pt" width="292">see Anal Biochem. 1985 Jul;148(1):254-9.</td>
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			<td class="xl21" height="21" style="height:15.75pt">Syringe and Blunt Cannula</td>
			<td class="xl21">Monoject</td>
			<td class="xl21">8881513918 and 888202017</td>
			<td class="xl20"></td>
		</tr>
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			<td class="xl21" height="21" style="height:15.75pt">Wide-bore pipette tips</td>
			<td class="xl21">Fischerbrand</td>
			<td class="xl21">02-707-134</td>
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			<td class="xl21" height="21" style="height:15.75pt">Plaque counter</td>
			<td class="xl21">New Brunswik Scientific</td>
			<td class="xl21">Colony Counter Model C-110</td>
			<td class="xl20"></td>
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			<td class="xl21" height="21" style="height:15.75pt">Culture tubes</td>
			<td class="xl22" style="width:168pt" width="224">Fischerbrand</td>
			<td class="xl21">14-961-33</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="height:15.75pt">Cell-free system</td>
			<td class="xl22" style="width:168pt" width="224">Mycroarray Inc</td>
			<td class="xl22" style="width:219pt" width="292">Mytxtl</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:162pt;height:15.75pt" width="216">BioComp Gradient Master</td>
			<td class="xl17" style="width:168pt" width="224">BioComp Instruments</td>
			<td class="xl17" style="width:219pt" width="292">Model 105ME</td>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:162pt;height:15.75pt" width="216">LB agar plate recipe</td>
			<td class="xl20"></td>
			<td class="xl20"></td>
			<td class="xl24" style="width:512pt" width="683">25 g/L Luria-Bertani medium (LB Broth, Miller - Fisher BioReagents product number BP1426) and 15 g/L Bacto-Agar solid (Brenton, Dickenson and Company - product number 214010).</td>
		</tr>
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synthesis of infectious bacteriophages in an e. coli-based cell-free expression system

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

We show four representative results. In Figure 1, we present a set of negative controls to ensure that the cell-free TXTL system and the phage DNA stocks are not contaminated with living E. coli cells. We verify that the cell-free TXTL system is free of intact E. coli cell contamination by plating both a non-incubated and incubated reaction solution void of genomic DNA (Figure 1A and <strong cla...

Watch this Scientific Journal Video about Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System at JoVE.com

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

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

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

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

The overall goal of this experiment is to describe how bacteriophages are synthesized from their genome using an all E.coli cell-free transcription translation system. This method can help answer key questions in the synthetic biology field, such as elucidating the molecular self-assembly mechanisms in living systems. The main advantage of this technique is the versatility of the platform.Many biological systems can be fully recapitulated and finely tuned for experimental analysis. Begin this procedure with preparation of phage plaques as described in the text protocol. Use the end of a sterile Pasteur pipette to core and remove a single plaque.Blow the plaque into the diluted cells. Then incubate at 37 degrees Celsius for two hours. To test for full infection, take a 500 microliter sample into a 1.5 milliliter tube and add 10 microliters of chloroform.Immediately vortex the mixture at high speed and observe if the solution clarifies, which indicates phage infection. Once the cells are fully infected, incubate for another two hours. Next, add DNase before centrifuging the cells at 8, 000 times g and four degrees Celsius.Discard the supernatant, then re-suspend the pellet in 10 milliliters of 1X Tris magnesium chloride. Add 500 microliters of chloroform and vortex at high speed to lyse the cells. Clarify by centrifugation at 12, 000 times g for 10 minutes at four degrees Celsius.Then decamp the supernatant into a 15 milliliter conical tube and store at four degrees Celsius. Prepare four five to 45%sucrose gradients by pipetting 2.5 milliliters of 45%sucrose into four ultracentrifuge tubes. Top the layer with approximately 2.6 milliliters of 5%sucrose, stopping when the liquid reaches two millimeters from the rim of the tube.Mix the gradients by tilt tube rotation using a gradient forming instrument for 43 seconds at 86 degrees and 23 RPM. Using a one milliliter pipette, add 500 microliters of the pooled phage suspension by bringing the tip into contact with the liquid surface and very slowly dispensing the volume onto the top of the sucrose gradient, being careful to not mix through rapid pipetting. Centrifuge at 70, 000 times g for 20 minutes at four degrees Celsius.Submerge a blunt canula into the solution until the tip is centered on the very upper edge of the phage band. Remove the band by drawing on the syringe plunger until the majority of the phage band has been removed. Then add the sucrose volume with the suspended phage to three or four ultracentrifuge tubes.Fill the tubes to between three and four millimeters from the top of the tube with cold 1XTM. Cover the tubes with paraffin film and invert to mix. Place the tubes back in an ultracentrifuge and spend at 145, 000 times g for one hour at four degrees Celsius.Quickly pour off the supernatant and drain upside down on disposable wipes. Divide 200 to 400 microliters of cold 1XTM across all pellets and re-suspend overnight at four degrees Celsius. No shaking is required.The day before making titers, prepare a culture of E.coli host cells in 10 milliliters of LB growth media and leave the culture in a shaking incubator set to 250 RPM and 37 degrees Celsius overnight. Next, prepare a master mix by pipetting 5.25 milliliters of top agar, 220 microliters of diluted phage samples, and 50 microliters of host bacterial cells to a 14 milliliter culture tube. Cap the culture tube and vortex at high speed.Retrieve two culture plates from the 37 degree Celsius incubator. Then add 2.5 milliliters of master mix slowly to the center of the first plate. Gently rotate the plate by hand to distribute the master mix evenly so that it spans the entire culture plate.Wait 20 minutes to ensure solidification of the top agar. Incubate the plates at 37 degrees Celsius for four to seven hours. Count the plaques and determine the phage concentration for each reaction sample.Dilute a portion of the phage stock to 400 microliters with 1XTM buffer in a fresh 1.5 milliliter tube. Add an equal volume of Tris phenol chloroform to the dilution. Shake the mixture gently on a laboratory rocker or by hand for five minutes.Then centrifuge the resultant emulsion at 17, 000 times g in a benchtop centrifuge for five to 10 minutes. Remove the upper aqueous phase to a new tube, taking care not to disturb the interphase boundary. Repeat these steps an additional two times for a total of three phenol extractions.Transfer the aqueous phase to a fresh tube and add an equal volume of chloroform. Shake and centrifuge before transferring the aqueous DNA sample to a clean tube. Next, add 0.4 volumes of three molar sodium acetate and three volumes of 95%ethanol.Place the tube in a minus 20 degree Celsius freezer for overnight precipitation. Centrifuge at 17, 000 times g in a benchtop centrifuge for 10 to 15 minutes. Remove and discard the supernatant, either by decanting or pipetting.Then add 500 microliters of 70%ethanol and gently shake the tube until the pellet floats free from the bottom of the tube. Following centrifugation, remove and discard the ethanol, taking care to not disturb the pellet. After adding ethanol and centrifuging as before, remove and discard the ethanol a second time, once again taking care not to disturb the pellet.Air dry the pellet on the benchtop for 30 to 60 minutes. Once dry, add 50 microliters of double-distilled water to the pellet and incubate for one hour at room temperature or overnight at four degrees Celsius to re-suspend. Determine the DNA concentration using absorption measurements at 280 nanometers.To perform the cell-free reaction, aliquot the indicated volume of crude extract, 33%of the final reaction volume, into a microcentrifuge tube. Prepare the master mix as listed in the text protocol. Add the appropriate volume of each component to the crude extract.After adding the last component, homogenize the reaction by vortexing. Then split the master mix into n microcentrifuge tubes. Next, add the indicated volumes of the variable components to the master mix array.Add water to each reaction to reach the desired final reaction volume before vortexing. Then incubate the microcentrifuge tubes at 29 degrees Celsius for at least eight hours or overnight. After preparing the cells for phage titer as described in the text protocol, titer the final cell-free reaction LB solution as demonstrated earlier in this video.Shown here are representative results of a cell-free reaction synthesizing phi X 174 phage. This phage plaque shows a successful phage reaction that was titered at an appropriate dilution, evident by the countable number of plaques. However, a successful reaction can show up poorly if the dilution factor is too low for the phage yield of the reaction.This plate shows an uncountable number of plaques. Shown here is a plate with inhomogeneous distribution of phage plaques due to insufficient pre-equilibration of culture plate temperature. During the phage titer, the culture plate solidified the reaction in the lower right portion of the plate.Molecular crowding conditions have a strong effect on phage synthesis for phi X 174 and T7.In particular, phage yield for T7 drops by three orders of magnitude when PEG concentrations drop from four to 2%weight by volume. Once mastered, phage purification and DNA extraction can be done in two days if it is performed properly. The cell-free reaction takes just hours when there are no issues.After watching this video, you should have a good understanding of how to grow phage, extract and purify their DNA, and use the result to reconstitute fully infectious phage using the cell-free reaction system. Working with phenol can be extremely hazardous, and precautions such as lab coat, eye protection, and gloves should be worn while performing this procedure. All work with open containers of phenol should be performed in a fume hood.

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Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

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

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

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

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

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 ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is ...

OnePot PURE Cell-Free System

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic biology. Unfortunately, commercially available systems are costly, and their tunability is limited. In comparison, a home-made approach can be customized based on user needs. However, the preparation of home-made systems is time-consuming and arduous due to the need for ribosomes as well as 36 medium scale protein purifications. Streamlining protein purification by coculturing and co-purification allows for minimizing time and labor requirements. Here, we present an easy, adjustable, time- and cost-effective method to produce all PURE system components within 1 week, using standard laboratory equipment. Moreover, the performance of the OnePot PURE is comparable to commercially available systems. The OnePot PURE preparation method expands the accessibility of the PURE system to more laboratories due to its simplicity and cost-effectiveness.

We present a fast and cost-effective method to produce the recombinant PURE cell-free TX-TL system using standard laboratory equipment.

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic ...

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.

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

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

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

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