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.).

<ol>
	<li>Carlson, E. D., Gan, R., Hodgman, C. E., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+protein+synthesis:+Applications+come+of+age.">Cell-free protein synthesis: Applications come of age.</a> Biotechnol Adv. , (2011).</li><li>Hodgman, C. E., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+synthetic+biology:+Thinking+outside+the+cell.">Cell-free synthetic biology: Thinking outside the cell.</a> Metab Eng. , (2011).</li><li>Caschera, F., 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=Synthesis+of+2.3+mg/mL+of+protein+with+an+all+Escherichia+coli+cell-free+transcription-translation+system.">Synthesis of 2.3 mg/mL of protein with an all Escherichia coli cell-free transcription-translation system.</a> Biochimie. 99, 162-168 (2014).</li><li>Shin, J., 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=An+E.+coli+cell-free+expression+toolbox:+application+to+synthetic+gene+circuits+and+artificial+cells.">An E. coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells.</a> ACS Synth Biol. 1 (1), 29-41 (2012).</li><li>Chemla, Y., Ozer, E., Schlesinger, O., Noireaux, V., Alfonta, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+expanded+cell-free+protein+synthesis+using+endogenous+pyrrolysyl+orthogonal+translation+system.">Genetically expanded cell-free protein synthesis using endogenous pyrrolysyl orthogonal translation system.</a> Biotechnol Bioeng. , (2015).</li><li>Hong, S. H., Kwon, Y. C., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-standard+amino+acid+incorporation+into+proteins+using+Escherichia+coli+cell-free+protein+synthesis.">Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis.</a> Frontiers in Chemistry. 2, (2014).</li><li>Noireaux, V., Bar-Ziv, R., Libchaber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+cell-free+genetic+circuit+assembly.">Principles of cell-free genetic circuit assembly.</a> Proc Natl Acad Sci U S A. 100 (22), 12672-12677 (2003).</li><li>Sun, Z. Z., Yeung, E., Hayes, C. A., Noireaux, V., Murray, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+DNA+for+Rapid+Prototyping+of+Synthetic+Biological+Circuits+in+an+Escherichia+coli+Based+TX-TL+Cell-Free+System.">Linear DNA for Rapid Prototyping of Synthetic Biological Circuits in an Escherichia coli Based TX-TL Cell-Free System.</a> Acs Synthetic Biology. 3 (6), 387-397 (2014).</li><li>Takahashi, M. 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. H., 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=Coarse-grained+dynamics+of+protein+synthesis+in+a+cell-free+system.">Coarse-grained dynamics of protein synthesis in a cell-free system.</a> Phys Rev Lett. 106 (4), 048104 (2011).</li><li>Daube, S. S., Arad, T., Bar-Ziv, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+co-synthesis+of+protein+nanoassemblies:+tubes,+rings,+and+doughnuts.">Cell-free co-synthesis of protein nanoassemblies: tubes, rings, and doughnuts.</a> Nano Lett. 7 (3), 638-641 (2007).</li><li>Chen, X., 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=An+immunoblot+assay+reveals+that+bacteriophage+T4+thymidylate+synthase+and+dihydrofolate+reductase+are+not+virion+proteins.">An immunoblot assay reveals that bacteriophage T4 thymidylate synthase and dihydrofolate reductase are not virion proteins.</a> J Virol. 69 (4), 2119-2125 (1995).</li><li>Sun, Z. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+implementing+an+Escherichia+coli+based+TX-TL+cell-free+expression+system+for+synthetic+biology.">Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology.</a> J Vis Exp. (79), e50762 (2013).</li><li>Shin, J. N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+cell-free+expression+with+the+endogenous+E.+coli+RNA+polymerase+and+sigma+factor+70.">Efficient cell-free expression with the endogenous E. coli RNA polymerase and sigma factor 70.</a> J Biol Eng. , (2010).</li><li>Caschera, F., 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=Preparation+of+amino+acid+mixtures+for+cell-free+expression+systems.">Preparation of amino acid mixtures for cell-free expression systems.</a> Biotechniques. 58 (1), 40-43 (2015).</li><li>Minton, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+can+biochemical+reactions+within+cells+differ+from+those+in+test+tubes.">How can biochemical reactions within cells differ from those in test tubes.</a> J Cell Sci. 119, 2863-2869 (2006).</li><li>Minton, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+macromolecular+crowding+for+protein+assembly.">Implications of macromolecular crowding for protein assembly.</a> Curr Opin Struct Biol. 10 (1), 34-39 (2000).</li><li>Zimmerman, S. B., Minton, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+crowding:+biochemical,+biophysical,+and+physiological+consequences.">Macromolecular crowding: biochemical, biophysical, and physiological consequences.</a> Annu Rev Biophys Biomol Struct. 22, 27-65 (1993).</li></ol>

The authors declare the following competing financial interest(s): Noireaux laboratory receives research funds from MYcroarray, a distributor of the MYtxtl cell-free protein expression kit.

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 the phage numbers to rapidly outstrip the cell count, allowing for multiple copies of phage in each host cell. This forces the cell into a quasi-stable state where it will not undergo lysis. If this state is not reached, the cells will undergo cascading, confluent lysis, and release their viral load. If superinfection cannot be reached using the above initial viral concentrations, lower the initial multiplicity of infection by a factor of ten before making a subsequent attempt. If the host cells do lyse before harvest and superinfection is not reached, proceed immediately with a separate purification procedure. Add DNase I to 5 &#181;g/mL and 500 &#181;L CHCl3 to finalize all cellular lysis. Continue to swirl for an additional 5 min and then centrifuge at 10,000 x g to remove cellular debris. Decant and centrifuge the supernatant at 75,000 x g for 1-2 h to pellet the phage and resuspend overnight in 1 mL total 1x TM buffer at 4 &#176;C without shaking. When testing for superinfection using a small volume of cells, it is possible to gauge how close cells are to lysis by how quickly they lyse when exposed to the added chloroform. If the cells lyse and the solution clarifies almost instantly, the cell walls are very unstable and cells should be harvested immediately to prevent large-scale cascade lysis. If the test lyses within 1-3 min, the larger volumes may continue to superinfect for up to 2 h, depending on the local strain of E. coli in use. If the cells do not lyse within 5 min, phage growth has not caught up to the E. coli growth and primary incubation should continue. Perform the test again in 30-60 min.
When purifying phage on a prepared sucrose gradient, the phage will appear as a very thick, pearlescent band between one third and two thirds of the way down the tube. Assuming a relatively successful lysis and cellular remnant purification step, there may be other, light bands but none of similar density or size. The large, opaque band contains the phage and is to be removed from the solution with a sterile syringe and blunt cannula.
When starting the third section of the protocol, the cell-free reaction and phage titer, it is best to use fresh culture plates (less than one week old) for the host bacteria plate (from step 3.5) and also the culture plates used for the titer analysis (from step 3.21). This will promote stronger growth of host bacteria cells and higher infection efficiency for the phage titer. Phage culture plates must be pre-incubated at 37 &#176;C for a minimum of 1 h before starting the phage titer portion of the protocol. Without pre-incubation of the phage culture plates, the top-agar solution (containing the synthesized phage and host cell culture) will not homogeneously distribute across the surface of the culture plate. Instead, the top-agar solution will homogeneously solidify on the surface (Figure 2b). This increases the probability of multiple phages localizing in the same spot, which can cause an underestimate of phage yield in the cell-free reaction.
Each plaque represents the event of one synthesized phage infecting a host cell, replicating within the host cell, and lysing the host cell. Plaques grow in size during the incubation period from the progeny phage infecting neighbor host cells from the initial infection event. If no plaques are observed, it is possible that the dilution factor made in Protocol section 4.6 is too high resulting in a low probability that any synthesized phage infects a host cell. To remedy this, decrease the dilution factor by 3-4 orders of magnitude (or as necessary) and repeat the titer experiment. Conversely, the dilution factor could be too high resulting in plaques spanning the entire surface of the culture plate, making it impossible to count the phage yield. In this case, increase the dilution factor by 3-4 orders of magnitude (or as necessary). For a well-characterized phage, the yield of the cell-free reaction will be known and only one dilution factor per reaction point will be necessary for the titer experiment. When characterizing a new phage, it is best to include 2-3 different dilution factors per reaction point, ensuring that one of the dilutions will yield a countable plaque (see Figure 3 for differences between a countable and uncountable number of plaques on a plate).
If no plaque is observed after modifying the dilution factor of the phage titer, this could indicate that the cell-free reaction was unsuccessful in that gene expression did not occur. A possible explanation for this could be structural damage of reagents/phage DNA due to the homogenization of the cell-free reaction by vortexing. To remedy this issue, repeat the reaction and homogenize by gently tapping the reaction tube five to seven times with a finger, or by slowly mixing the reaction with a pipette five to seven times.
The cell-free reaction system originates from E. coli and endogenously contains both E. coli RNA polymerase and the primary sigma factor, sigma 70, which combine to form the holoenzyme necessary to recognize the main promoter consensus sequences of E. coli genes. Additionally, the cell-free reaction system can recapitulate the entire sigma factor transcription scheme by exogenously supplying six other E. coli sigma factor genes under the promoter consensus sequence of sigma 70. This allows the capability to synthesize all bacteriophages whose genomes are compatible with E. coli&#39;s transcription/translation machinery. This leaves out a subset of bacteriophage that cannot be studied or synthesized with the cell-free reaction system utilized here.
The cell-free transcription/translation platform allows for an impressive level of control and flexibility of reaction conditions compared to in vivo methods of study. We can finely tune many biochemical and genetic components of a phage reaction and directly observe the effects in less than 24 h, as shown in Figure 4 for molecular crowding. The effects of molecular crowders, such as PEG, on the self-assembly of macromolecular complexes have been theoretically described and demonstrated in vitro18,19,20. Molecular crowding is an entropy driven mechanism that increases the association constant of large biomolecular structures. The cell-free approach allows the researcher to probe complicated biological processes like the biophysics of self-assembly with model systems, like bacteriophages, without the intrinsic limitations of in vivo work.
This platform also allows for the investigation of the utility of phages in applied fields. Components of phage genomes can be repurposed and quickly tested in a cell-free reaction system with the goal of incorporating results to novel nanostructures. Bacteriophages can be modified in order to increase the selectivity of host cell infection, which would promote the utility of phage therapy as an alternative for antibiotic-resistant bacteria.

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).

<table width="899">
	<colgroup>
		<col style="width:162pt" width="216" />
		<col style="width:168pt" width="224" />
		<col style="width:219pt" width="292" />
		<col style="width:125pt" width="167" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<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>
		<tr height="21" style="height:15.75pt">
			<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>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<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>
		<tr height="21" style="height:15.75pt">
			<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>
			<td class="xl20"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<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>
		</tr>
		<tr height="21" style="height:15.75pt">
			<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>
	</tbody>
</table>

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 Figure 1B). If the cell-free TXTL system was contaminated with E. coli cells, growth would be observed on the plate. Furthermore, we verify that the cell-free reaction, and therefore not any possible contaminant cells, are responsible for the synthesis of phage by plating non-incubated and incubated reactions with the phage genome (Figure 1C and Figure 1D). The plate with the non-incubated reaction (Figure 1C) shows zero plaque while the plate with the incubated reaction (Figure 1D) shows plaque, indicating the cell-free TXTL system was responsible for phage synthesis.
In Figure 2, we compare a homogeneous versus inhomogeneous plaque assay. Figure 2B illustrates a gradient of plaque density across the culture plate, which is sub-optimal in comparison to a homogeneous plaque density of the plate in Figure 2A. The source of this observed result is from the rapid cooling of master mix of top agar, sample and host bacterial cells when dispensed onto a culture plate during the phage titer portion of the experiment. To avoid an inhomogeneous spread of phage plaques, ensure that the culture plates are equilibrated to 37 &#176;C before starting the phage titer experiment.
A critical step to properly count phages is to dilute the phage solution sufficiently to obtain between 50 and 200 plaques per plate (Figure 3). Below this range, it is difficult to determine true statistics, and above this range, the plaques overlap, preventing accurate counts. The number of phages synthesized in TXTL depends on the biochemical settings, such as the concentration of salts (potassium and magnesium), of molecular crowders, and of genomes. In Figure 4, we show a few examples of tests for the phages T7 and &#934;&#935;174. For a phage that replicates its DNA, such as T7, we observe a high efficiency of phage synthesis in the cell-free reaction: three infectious phages are synthesized per genome molecule in the reaction. One can expect a lower efficiency of reaction for a phage that does not replicate its DNA, such as &#934;&#935;174: the ratio of synthesized infectious phage to genome molecule is 1:5.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56144/56144fig1.jpg" /> 
Figure 1: Plaque assay controls and successful infectious phage production using a cell-free reaction (CFR) system. (A) No genome was added and no incubation (0 min as incubation time) of CFR was performed. The reaction was then plated without host E. coli. Result shows no viable host cell contamination of CFR. (B) No genome was added and CFR was incubated 12 h at 29 &#176;C and plated without host E. coli. Results show no viable host cell contamination of CFR, even after incubation. (C) 1 nM genome included with no incubation of CFR (0 min as incubation time) and plated with host E. coli. Result shows no phage contamination of the genome solution. (D) 1 nM genome included and CFR incubated 12 h at 29 &#176;C. and plated with host E. coli. Results show successful infectious phage replication in CFR. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56144/56144fig1large.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/56144/56144fig2.jpg" /> 
Figure 2: Homogeneous versus inhomogeneous spread of phage reaction during titer experiment. A possible effect of performing a phage titer on culture plates not equilibrated to a temperature of 37 &#176;C is the inhomogeneous spread of phage reaction across the culture plate. A good result displaying a homogeneous spread of phage reaction is shown in (A), where the plaques are evenly distributed across the surface of the plate. A sub-optimal result is shown in (B), where the plaques are concentrated in the lower-left portion of the culture plate. This can occur with the top-agar, phage reaction, and host cell master mix are dispensed onto a culture plate that is not at a temperature of 37 &#176;C. The majority of the master mix solidifies in the lower-left region, and a homogeneous spread is not possible. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56144/56144fig2large.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/56144/56144fig3.jpg" /> 
Figure 3: Setting an appropriate dilution factor for titers of a cell-free phage reaction. (A) This plate displays a reasonably countable number of plaques. The dilution factor used for the titer portion of the experiment diluted the yield of the phage reaction enough to leave a countable number of plaques on the plate. Conversely in (B) the dilution factor was too low with respect to the yield of synthesized phage, and needs to be adjusted. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56144/56144fig3large.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/56144/56144fig4.jpg" /> 
Figure 4: Characterization of the cell-free synthesis of phages &#934;&#935;174 and T7. (A) Number of synthesized &#934;&#935;174 and T7 phages (PFU/mL: plaque forming units per milliliter) as a function of PEG 8000 concentration in the cell-free reaction, measured after 16 h of incubation at 29 &#176;C. (B) Number of synthesized &#934;&#935;174 and T7 phages as a function of magnesium-glutamate concentration in the cell-free reaction, measured after 16 h of incubation at 29 &#176;C. (C) Number of synthesized &#934;&#935;174 phage as a function of &#934;&#935;174 DNA genome concentration in the cell-free reaction, measured after 16 h of incubation at 29 &#176;C. (D) Number of synthesized T7 phage as a function of T7 DNA genome concentration in the cell-free reaction, measured after 16 h of incubation at 29 &#176;C. All error bars displayed are standard deviations of three repeated trials. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56144/56144fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/56144/56144table1.jpg" /> 
Table 1: Reaction composition. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56144/56144table1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

synthesis-infectious-bacteriophages-an-e-coli-based-cell-free

Nanyang Technological University

Research

JoVE Journal

Bioengineering

14.5K Views.  University of Minnesota. 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, ΦΧ174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

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

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 ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

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.