This work was supported by JSPS KAKENHI grant number JP18K14260 and the budget of Japan Agency for Marine-Earth Science and Technology. We thank Shigeru Deguchi (JAMSTEC) and Tetsuro Ikuta (JAMSTEC) for providing the characterization facilities. We thank Ken Takai (JAMSTEC) for commercial software support. The microfabrication was conducted at Takeda Sentanchi Supercleanroom, The University of Tokyo, supported by &#34;Nanotechnology Platform Program&#34; of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, Grant Number JPMXP09F19UT0087.

The authors have nothing to disclose.

The highly quantitative measurement based on the highly uniform, stable, and biocompatible droplets in FemDA enabled the discrete distribution, the unique feature of our study differing from others. We systematically optimized and detailed the microfabrication and droplet formation processes in this paper. There are several critical steps in the established protocol.
First, the uniform coating of highly viscous CYTOP polymer on the rectangular thin glass substrate largely determines the qualit...

Researchers use reactors to carry out bio/chemical reactions. There are significant efforts that have been made to reduce the size of the reactor and increase the experimental throughput in order to lower the reagent consumption while improving the work efficiency. Both aspects aim to liberate researchers from a heavy workload, decreasing the cost and speeding up research and development. We have a clear historical roadmap about the development of the reactor technologies from the viewpoint of reaction volumes and throughput: single beakers/flask/test-tubes, milliliter tubes, microliter tubes, microliter 8-tube strips, microliter 96/384/1536-well plate, and microfluidic nanoliter/picoliter/femtoliter reactors1,2,3,4,5,6,7. Analogous to downsizing the feature size of transistors on integrated circuit chips in the semiconductor industry in the past decades, bio/chemical microreactors are going through volume reduction and system integration. Such small-scale tools have had a profound impact on cell-based or cell-free synthetic biology, biomanufacturing, and high-throughput prototyping and screening8,9,10,11,12. This paper describes our recent effort on the development of a unique droplet array technology and demonstrates its application in CFPS13, a fundamental technology for synthetic biology and molecular screening communities14. In particular, we intentionally provide an optimized and low-cost protocol to make the FemDA device accessible to everyone. The low-cost and easy-to-handle protocol for the miniaturized device would contribute to the educational purposes of universities and help spread the technology.
FemDA prepares femtoliter droplets at an ultrahigh density of 106 per 1 cm2 on a planar glass substrate. We coated a hydrophobic polymer, CYTOP15, on the glass substrate and selectively etched (removed) CYTOP at predefined positions to generate a microchamber array on the substrate. Thus, the resulting microchamber is composed of a hydrophobic sidewall (CYTOP) and a hydrophilic bottom (glass). When sequentially flowing water and oil over the patterned surface, the water can be trapped and sealed into the microchambers. The hydrophilic-in-hydrophobic structure is vital for repelling water outside the microchambers, isolating individual microreactors, and retaining a tiny aqueous solution inside the femtoliter space. The unique property was successfully applied for the preparation of water-in-oil droplets and lipid bilayer microcompartments16,17. Compared to the prototype device16, we first optimized the microfabrication process to realize a complete removal of the CYTOP polymer as well as a full exposure of the glass bottom. CYTOP is a special fluoropolymer featuring extremely low surface tension (19 mN/m) lower than that of conventional microreactor materials such as glass, plastics, and silicone. Its good optical, electrical, and chemical performance have already been utilized in surface treatment of microfluidic devices18,19,20,21,22,23,24. In the FemDA system, to achieve good wetting of the oil on the CYTOP surface, the surface tension of the oil must be lower than that of the solid surface25. Otherwise, the liquid oil in contact with the solid surface tends to become spherical rather than spreading over the surface. Besides, we found that some popular perfluorocarbon oils (e.g., 3M FC-40)16 and hydrofluoroether oils (e.g., 3M Novec series) can dissolve CYTOP as a result of the amorphous morphology of CYTOP, which is fatal to quantitative measurement and would be questionable in terms of cross-contamination among droplets. Fortunately, we identified a biocompatible and environmentally friendly oil exhibiting lower (&#60; 19 mN/m) surface tension13. We also found a new surfactant that can dissolve in the selected oil and function in a low concentration (0.1%, at least 10-fold lower than previously reported popular ones26,27)13. The resulting water/oil interface can be stabilized by the surfactant. Because of the high evaporation rate of the oil, following the flush with the oil, we applied another biocompatible and environmentally friendly oil to replace the first one to seal the microchambers. We call the first oil (ASAHIKLIN&#160;AE-3000 with 0.1 wt % SURFLON S-386) the &#8220;flush oil&#8221; and the second oil (Fomblin Y25) the &#8220;sealing oil,&#8221; respectively.
The two-step oil-sealing strategy can realize a robust formation of the femtoliter droplet array within minutes and without sophisticated instrumentation. Due to the evaporation problem, it has been considered challenging to generate microreactors smaller than picoliter volumes28. FemDA addressed this issue by systematically optimizing the materials and processes used for the preparation of microreactors/droplets. Several noteworthy features of the resulting droplets include the high uniformity (or monodispersity), stability, and biocompatibility at the femtoliter scale. The coefficient of variation (CV) of the droplet volume is only 3% (without vignetting correction for the microscopic images), the smallest CV among droplet platforms in the world, which ensures a highly parallel and quantitative measurement. The femtoliter droplet is stable for at least 24 hours without cross-contamination among droplets at room temperature, which is valuable for a reliable time-course measurement. Regarding the biocompatibility, we succeeded in synthesizing various proteins from a single-copy template DNA in the femtoliter droplet, which had previously been considered difficult or inefficient29,30. It would be worthy of elucidating why some proteins capable of being synthesized in the FemDA cannot be synthesized in other droplet systems. FemDA was not merely a technical advancement, but also realized an unprecedentedly quantitative measurement that can correlate the protein yield (as reflected by the fluorescence intensity of the droplet) to the number of template DNA molecules in each droplet. As a result, the histogram of the fluorescence intensity of droplets from FemDA-based CFPS showed a discrete distribution that can be nicely fitted by a sum of Gaussian distributions of equal peak-to-peak intervals. Moreover, the probability of occurrence of droplets containing different numbers of DNA molecules was a perfect fit to a Poisson distribution31. Thus, the different protein yield in each droplet can be normalized based on the discrete distribution. This critical feature allows us to separate the enzymatic activity information from the apparent intensity, that has not been available with other microreactor platforms yet. Existing microfluidic cell/droplet sorting systems are skilled in fully automatic sorting and good at concentrating samples but sometimes can only output a relatively broad or long-tailed histogram in the analytical aspect32,33. Our highly quantitative and biocompatible FemDA system sets a new benchmark and a high analytical standard in the field of microreactor development.
The oils and surfactants that could be used for the preparation of droplets are still very limited34. The combination of ASAHIKLIN AE-3000 and SURFLON S-386 established in FemDA is a new member of the growing arsenal of the physiochemical interface between the aqueous phase and the oil phase13. The new interface in FemDA is physically stable, chemically inert, and biologically compatible with the complex transcription, translation, and post-translational modification machinery for many sorts of proteins13. It would be rather attractive to find a protein that cannot be synthesized in the droplet settings instead. Besides, the cost saving of reagents is more evident in the femtoliter droplet system than that in nanoliter and picoliter reactor systems35,36. In particular, there would often be a large dead volume, which is mainly caused by tubing or external supplies, in microfluidic droplet generation systems but not in our FemDA. The array format is also favored by repeated and detailed microscopic characterization (similar to so-called high-content analysis) for every single reactor37, rather than only a single snapshot for a fast-moving object. The femtoliter scale enabled the integration of over one million reactors on a finger-sized area, while the same number of nanoliter reactors (if it exists) requires over square meter area, which would be undoubtedly impractical to manufacture or use such system.

<table><tbody><tr><td>(3-aminopropyl)triethoxysilane</td><td>Sigma-Aldrich</td><td>440140</td><td></td></tr><tr><td>1 mL syringe</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>2-propanol</td><td>Kanto Chemical</td><td>EL grade</td><td>EL: for electronic use.</td></tr><tr><td>3D laser scanning confocal microscope</td><td>Lasertec</td><td>OPTELICS HYBRID</td><td>Other similar microscopes (e.g., Keyence VK-X1000, Olympus LEXT OLS5000) are also applicable.</td></tr><tr><td>50 mL syringe</td><td>Terumo</td><td>SS-50LZ</td><td></td></tr><tr><td>6,8-difluoro-4-methylumbelliferyl phosphate</td><td>Thermo Fisher Scientific</td><td>D6567</td><td>Prepare a 5 mM stock solution in dimethyl sulfoxide</td></tr><tr><td>Acetone</td><td>Kanto Chemical</td><td>EL grade</td><td>EL: for electronic use.&lt;br/&gt; Purity 99.8%.</td></tr><tr><td>Air blower</td><td>Hozan</td><td>Z-263</td><td></td></tr><tr><td>Aluminum block</td><td>BIO-BIK</td><td>AB-24M-02</td><td></td></tr><tr><td>Aluminum microtube stand</td><td>BIO-BIK</td><td>AB-136C</td><td></td></tr><tr><td>ASAHIKLIN AE-3000</td><td>AGC</td><td>(Test sample)</td><td>Free test sample may be available upon inquiry to AGC.</td></tr><tr><td>BEMCOT PS-2 wiper</td><td>Ozu</td><td>028208</td><td></td></tr><tr><td>Biopsy punch with plunger</td><td>Kai</td><td>BPP-10F</td><td></td></tr><tr><td>Cover glass</td><td>Matsunami Glass</td><td>No. 1 (24 mm &amp;times; 32 mm, 0.13~0.17 mm thickness)</td><td>Size-customized.</td></tr><tr><td>Cover glass staining rack</td><td>Nakayama</td><td>803-131-11</td><td></td></tr><tr><td>CRECIA TechnoWipe clean wiper</td><td>Nippon Paper Crecia</td><td>C100-M</td><td></td></tr><tr><td>Cutting mat</td><td>GE Healthcare</td><td>WB100020</td><td></td></tr><tr><td>CYTOP</td><td>AGC</td><td>CTL-816AP</td><td></td></tr><tr><td>Deaeration mixer</td><td>Thinky</td><td>AR-100</td><td></td></tr><tr><td>Desktop cutter</td><td>Roland</td><td>STIKA SV-8</td><td></td></tr><tr><td>Developer</td><td>AZ Electronic Materials</td><td>AZ 300 MIF</td><td>AZ Electronic Materials was now acquired by Merck.&lt;br/&gt; Other alkaline developers may be also applicable but should require optimization of development conditions (time, temperature, etc.)</td></tr><tr><td>Double-coated adhesive Kapton film tape</td><td>Teraoka Seisakusho</td><td>7602 #25</td><td></td></tr><tr><td>Ethanol</td><td>Kanto Chemical</td><td>EL grade</td><td>EL: for electronic use.&lt;br/&gt; Purity 99.5%.</td></tr><tr><td>Fiji</td><td></td><td></td><td>Version: ImageJ 1.51n</td></tr><tr><td>Flat-cable cutter</td><td>Tokyo-IDEAL</td><td>MT-0100</td><td></td></tr><tr><td>Fomblin oil</td><td>Solvay</td><td>Y25, or Y25/6</td><td>Free test sample may be available upon inquiry to Solvay. Fomblin Y25/6 is an alternative if Y25 is not readily available.</td></tr><tr><td>Hot plate</td><td>AS ONE</td><td>TH-900</td><td></td></tr><tr><td>Injection needle</td><td>Terumo</td><td>NN-2270C</td><td>22G &amp;times; 70 mm</td></tr><tr><td>Inverted fluorescence microscope</td><td>Nikon</td><td>Eclipse Ti-E</td><td>Epifluorescence specification, CCD or sCMOS camera, motorized stage, autofocus system, and high NA objective lens are required.</td></tr><tr><td>KaleidaGraph</td><td>Synergy</td><td></td><td>Version: 4.5</td></tr><tr><td>Mask aligner</td><td>SUSS</td><td>MA-6</td><td>Other mask aligners are also applicable as long as the vacuum contact mode is avaliable.</td></tr><tr><td>MICROMAN pipette</td><td>GILSON</td><td>E M250E</td><td>Capillary piston tip: CP250</td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td></td><td>Version: 16.16.15</td></tr><tr><td>Mini vacuum chamber</td><td>AS ONE</td><td>MVP-100MV</td><td></td></tr><tr><td>Nuclease-free water</td><td>NIPPON GENE</td><td>316-90101</td><td></td></tr><tr><td>Parafilm</td><td>Amcor</td><td>PM-996</td><td></td></tr><tr><td>PCR tube</td><td>NIPPON Genetics</td><td>FG-021D/SP</td><td></td></tr><tr><td>Petri dish</td><td>AS ONE</td><td>GD90-15</td><td>Diameter 90 mm, height 15 mm.</td></tr><tr><td>Photoresist</td><td>AZ Electronic Materials</td><td>AZ P4903</td><td>AZ Electronic Materials was now acquired by Merck. AZ P4620 is an alternative.</td></tr><tr><td>Plate reader</td><td>BioTek</td><td>POWERSCAN HT</td><td></td></tr><tr><td>Polyethelene gloves</td><td>AS ONE</td><td>6-896-02</td><td>Trade name: Saniment.</td></tr><tr><td>PURExpress in vitro protein synthesis kit</td><td>New England Biolabs</td><td>E6800S or E6800L</td><td>For cell-free protein synthesis reaction.</td></tr><tr><td>Reactive-ion etching system</td><td>Samco</td><td>RIE-10NR</td><td>Other RIE systems are also applicable but should require optimization of RIE conditions (gas flow rate, chamber pressure, RF power, etching time, etc.)</td></tr><tr><td>RNase inhibitor</td><td>New England Biolabs</td><td>M0314S</td><td></td></tr><tr><td>Scotch tape</td><td>3M</td><td>810-1-18D</td><td></td></tr><tr><td>Sodium hydroxide solution</td><td>FUJIFILM Wako Pure Chemical</td><td>194-09575</td><td>8 M concentration; danger.</td></tr><tr><td>Spin coater</td><td>Oshigane</td><td>SC-308</td><td></td></tr><tr><td>SURFLON S-386 surfactant</td><td>AGC</td><td>(Test sample)</td><td>Free test sample may be available upon inquiry to AGC.</td></tr><tr><td>SYLGARD 184 silicone elastomer</td><td>Dow</td><td>Sylgard184</td><td>Chemical composition: polydimethylsiloxane. The default mixing ratio is base : curing agent = 10 : 1 (m/m).</td></tr><tr><td>Tweezers</td><td>Ideal-tek</td><td>2WF.SA.1&lt;br/&gt; 2A</td><td></td></tr><tr><td>Ultrasonic cleaner</td><td>AS ONE</td><td>ASU-2M</td><td></td></tr><tr><td>Vacuum chuck</td><td>Oshigane</td><td>(Customized)</td><td>Material: delrin; rectangular sample stage with multiple holes (48 holes, each with 1 mm diameter); the size is customzied to fit the size of the cover glass (24 mm &amp;times; 32 mm).</td></tr></tbody></table>

a femtoliter droplet array for massively parallel protein synthesis from single dna molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The microfabrication process consists of substrate cleaning, surface functionalization, CYTOP coating, photolithography, dry etching, photoresist stripping, and final cleaning. Importantly, the presented protocol allowed complete removal of the hydrophobic CYTOP polymer inside the microchambers Figure 3A), producing a highly parallel hydrophilic-in-hydrophobic structure on a standard cover glass substrate. With the aid of the oil sealing protocol, the uniform dimension of the resulting dropl...

Watch this Scientific Journal Video about A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules at JoVE.com

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and ...

a-femtoliter-droplet-array-for-massively-parallel-protein-synthesis

Universidad San Sebastian

Research

JoVE Journal

Chemistry

10.2K Views.  Japan Agency for Marine-Earth Science and Technology (JAMSTEC). The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

Video: A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

Cancer Research

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Bioengineering

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

Immunology and Infection

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...

Genetics

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

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

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

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

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

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

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

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

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

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

CFPS Protocols for Micro-Compartmentalized Synthetic Cells

Cell-Free Protein Synthesis System for Building Synthetic Cells

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.

Author Spotlight: Optimizing CFPS Systems for Synthetic Cell Construction

This protocol describes the Cell-Free Protein Synthesis (CFPS) system used in constructing synthetic cells. It outlines key stages with representative results in different micro-compartments. The protocol aims to establish best practices for diverse laboratories in the synthetic cell community, advancing progress in synthetic cell development.

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for ...

Biochemistry

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

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

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

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

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

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

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Cell-Free Protein Synthesis System for Building Synthetic Cells

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

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