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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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>
		<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>
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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 ...

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