Name: rapid, enzymatic methods for amplification of minimal, linear templates for protein prototyping using cell-free systems
Uploaded: 2021-06-14T14:30:00
Description: Watch this Scientific Journal Video about Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems at JoVE.com

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

1. Designing the gene fragment
NOTE: The gene fragment should have all the necessary genetic elements for transcription/translation, including promoter, ribosome binding site (RBS), start codon, the gene of interest, and terminator. While the terminator is not necessary for a linear expression template (LET), it will be important if the user decides to insert the sequence into a plasmid. These sequences were lifted from the pJL1-sfGFP plasmid55 (gift from Michael Jewett&#...

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

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

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

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

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

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

Watch this Scientific Journal Video about Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems at JoVE.com

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

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

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

Research

JoVE Journal

Bioengineering

Fluorescence detection methods for microfluidic droplet platforms

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

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

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

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

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

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

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

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

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

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

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

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

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 ...

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 ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder ...