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Cell-Free Biosensors: Synthetic Biology Without Borders
Abstract
Cell-free biosensors can take many forms and can range in complexity from single enzymes to engineered systems of biological components that support synthetic biology applications. This chapter will review the many recent innovations from this latter category and will explore how these more complex systems create synthetic networks to provide biosensors with signal amplification, programmability, high sensitivity, and even tolerance for analyte variation. In particular, cell-free biosensors that operate using isothermal amplification, coupled transcription and translation systems, and CRISPR-related mechanisms will be highlighted. Such DNA-/RNA-based technologies are an especially exciting category for cell-free biosensing, and here this rapidly evolving class of sensors, including toehold switch- and CRISPR-based systems, will be reviewed. Cell-free biosensors are also increasingly designed with companion hardware, and, in doing so, researchers are embedding the capacity for these otherwise laboratory-based reactions to be deployed in real-world applications. Among many innovations, this chapter will highlight how freeze-dried and paper-based systems, low-cost optical readers, and lateral flow devices are helping extend the reach of cell-free biosensors into new environments and applications. Taken together, the use of cell-free synthetic biology and engineered biochemical systems is an exciting category of biosensing and is on track to make significant contributions toward decentralizing the capacity for sensing.
... engineering, biological materials, environmental monitoring, medical diagnostics and therapeutic biomanufacturing (Garenne et al., 2021;Monck et al., 2022;Swartz, 2018;Zawada et al., 2022). Cell-free synthetic biology may also be poised to enable a radical biomanufacturing paradigm, whereby therapeutic and medical diagnostic technologies can be readily manufactured locally from rapidly designed and in silico distributed genetic sequences (Ogonah et al., 2017;Tinafar et al., 2022). Importantly, cell-free reactions can be lyophilised or silica dried and potentially employed in areas with limited cold chain access (Guzman-Chavez et al., 2022;Jaenes et al., 2022;Pardee, 2018;Pardee et al., 2014). ...
... This expands the utility of cell-free gene expression systems and has been posited as a route towards the on-demand production of cell-free-based diagnostics or therapeutics (Bundy et al., 2018;Guzman-Chavez et al., 2022;Jaenes et al., 2022;. Essentially, standardised cell-free reactions could conceivably be geographically distributed and then reconstituted locally, as required, with a customised genetic programme that encodes an appropriate biologic, vaccine or diagnostic genetic circuit (Ogonah et al., 2017;Pardee et al., 2016;Tinafar et al., 2022). It is conceivable that such approaches could be further developed to also incorporate cell-derived EVs or EV mimetics, whereby their inclusion might enable the targeted delivery of the cell-free produced therapeutic or add additional therapeutic modalities (e.g., theragnostics; Figure 2). ...
Abstract Extracellular vesicles (EVs) are lipid‐membrane nanoparticles that are shed or secreted by many different cell types. The EV research community has rapidly expanded in recent years and is leading efforts to deepen our understanding of EV biological functions in human physiology and pathology. These insights are also providing a foundation on which future EV‐based diagnostics and therapeutics are poised to positively impact human health. However, current limitations in our understanding of EV heterogeneity, cargo loading mechanisms and the nascent development of EV metrology are all areas that have been identified as important scientific challenges. The field of synthetic biology is also contending with the challenge of understanding biological complexity as it seeks to combine multidisciplinary scientific knowledge with engineering principles, to build useful and robust biotechnologies in a responsible manner. Within this context, cell‐free systems have emerged as a powerful suite of in vitro biotechnologies that can be employed to interrogate fundamental biological mechanisms, including the study of aspects of EV biogenesis, or to act as a platform technology for medical biosensors and therapeutic biomanufacturing. Cell‐free gene expression (CFE) systems also enable in vitro protein production, including membrane proteins, and could conceivably be exploited to rationally engineer, or manufacture, EVs loaded with bespoke molecular cargoes for use in foundational or translational EV research. Our pilot data herein, also demonstrates the feasibility of cell‐free EV engineering. In this perspective, we discuss the opportunities and challenges for accelerating EV research and healthcare applications with cell‐free synthetic biology.
... To date, a variety of organisms have been utilized to produce cell-free expression systems though E. coli-based extracts dominate the field owing to its wideness as the prototyping system in the lab. While the PURE system is more costly and labor-intensive to prepare due to the need of purification of more than 30 protein components, it may help increase the sensitivity for genetic biosensor [42,53,54]. Importantly, cell-free systems have great potential to be the platform for developing field-deployable biosensors by producing easy-to-interpret output either directly visible by the naked eye or by a smartphone with suitable excitation and emission filters placed in front of the camera or a portable 3D printed detector (Fig. 1f). ...
Wastewater surveillance is a powerful tool to understand community profiling in terms of health monitoring. Tracking biomarkers such as inorganic and organic pollutants, drugs, and pathogens in wastewater gives a general idea about the lifestyle and health status of a population as well as pollutant exposure caused by various toxic chemicals. Notably, tracing pathogenic clues could help predict and prevent disease outbreaks such as the ongoing COVID-19 pandemic in communities. To this end, developing portable biosensing platforms will facilitate the on-site monitoring of water contamination without requiring complex equipment. New technological developments in synthetic biology have advanced both synthetic gene circuit-based biosensors and new in vitro detection strategies coupled with easy-to-interpret visualization methods. Here, we summarize the latest advances in synthetic biology tools and discuss how they enable the development of rapid, low-cost, ease-to-use and field-deployable biosensors for monitoring a variety of water contaminants and health-related biomarkers in the environment.
... As the outbreak progressed, RT-qPCR fieldtesting laboratories closer to the front lines were established-however, challenges with testing capacity remained. The crisis left many wondering how many lives could have been saved if better de-centralized diagnostics were available earlier and, in many ways, was a catalyst for many of the field's first attempts at decentralizing molecular diagnostics [4]. Now, in the midst of COVID-19, we are facing a diagnostic crisis on a global scale. ...
The global spread of SARS-CoV-2 has shaken our health care and economic systems, prompting re-evaluation of long-held views on how best to deliver care. This is especially the case for our global diagnostic strategy. While current laboratory-based centralized RT-qPCR will continue to serve as a gold standard diagnostic into the foreseeable future, the shortcomings of our dependence on this method have been laid bare. It is now clear that a robust diagnostics pandemic response strategy, like any disaster planning, must include adaptive, diverse and decentralized solutions. Here we look at how the COVID-19 pandemic, and previous outbreaks, have set the stage for a new innovative phase in diagnostics and a rethinking of pandemic preparedness.
... The specific target probes designed by incorporating fluorophores and quenchers promise higher specificity and sensitivity in NA recognition. 127 As a result, combining mature amplification tools (such as RT-PCR, 128 LAMP, 114 and NASBA 129 ) and specific target probing technology is the predominant strategy for detecting viruses such as SARS-CoV and SARS-CoV-2. 11,130 For example, the detection of SARS-CoV by fluorescent detection has been reported with an LOD of 1 copy RNA per reaction by RT-PCR. ...
The pandemic of coronavirus disease 2019 (COVID-19) highlights the importance of rapid and sensitive diagnostics of viral infection that enables the efficient tracing of cases and the implementation of public health measures for disease containment. The immediate actions from both academia and industry have led to the development of many COVID-19 diagnostic systems that have secured fast-track regulatory approvals and have been serving our healthcare frontlines since the early stage of the pandemic. On diagnostic technologies, many of these clinically validated systems have significantly benefited from the recent advances in micro- and nanotechnologies in terms of platform design, analytical method, and system integration and miniaturization. The continued development of new diagnostic platforms integrating micro- and nanocomponents will address some of the shortcomings we have witnessed in the existing COVID-19 diagnostic systems. This Perspective reviews the previous and ongoing research efforts on developing integrated micro- and nanosystems for nucleic acid-based virus detection, and highlights promising technologies that could provide better solutions for the diagnosis of COVID-19 and other viral infectious diseases. With the summary and outlook of this rapidly evolving research field, we hope to inspire more research and development activities to better prepare our society for future public health crises.
Organisms from across the tree of life have evolved highly efficient mechanisms for sensing molecules of interest using biomolecular machinery that can in turn be quite valuable for the development of biosensors. However, purification of such machinery for use in in vitro biosensors is costly, while the use of whole cells as in vivo biosensors often leads to long sensor response times and unacceptable sensitivity to the chemical makeup of the sample. Cell-free expression systems overcome these weaknesses by removing the requirements associated with maintaining living sensor cells, allowing for increased function in toxic environments and rapid sensor readout at a production cost that is often more reasonable than purification. Here, we focus on the challenge of implementing cell-free protein expression systems that meet the stringent criteria required for them to serve as the basis for field-deployable biosensors. Fine-tuning expression to meet these requirements can be achieved through careful selection of the sensing and output elements, as well as through optimization of reaction conditions via tuning of DNA/RNA concentrations, lysate preparation methods, and buffer conditions. Through careful sensor engineering, cell-free systems can continue to be successfully used for the production of tightly regulated, rapidly expressing genetic circuits for biosensors.
Respiratory syncytial virus (RSV) infection is the most common clinical infectious disease threatening the safety of human life. Herein, we provided a sensitive and specific method for detection and differentiation of RSV subgroups A (RSVA) and B (RSVB) with colorimetric toehold switch sensors in a paper-based cell-free system. In this method, we applied the toehold switch, an RNA-based riboswitch, to regulate the translation level of β-galactosidase (lacZ) gene. In the presence of target trigger RNA, the toehold switch sensor was activated and the expressed LacZ hydrolyzed chromogenic substrates to produce a colorimetric result that can be observed directly with the naked eye in a cell-free system. In addition, nucleic acid sequence-based amplification (NASBA) was used to improve the sensitivity by amplifying target trigger RNAs. Under optimal conditions, our method produced a visible result for the detection of RSVA and RSVB with the detection limit of 52 aM and 91 aM, respectively. The cross-reaction of this method was validated with other closely related respiratory viruses, including human coronavirus HKU1 (HCoV-HKU1), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Furthermore, we used the paper-based carrier material that allows stable storage of our detection elements and rapid detection outside laboratory. In conclusion, this method can sensitively and specifically differentiate RSVA and RSVB and generate a visible colorimetric result without specialized operators and sophisticated equipment. Based on these advantages above, this method serves as a simple and portable detector in resource-poor areas and point-of-care testing (POCT) scenarios.
We demonstrate a simple, robust, and low-cost method for producing the PURE cell-free transcription-translation system. Our OnePot PURE system achieved a protein synthesis yield of 156 μg/mL at a cost of 0.09 USD/μL, leading to a 14-fold improvement in cost normalized protein synthesis yield over existing PURE systems. The OnePot method makes the PURE system easy to generate and allows it to be readily optimized and modified.
Several groups have recently reported on the utility of cell-free expression systems to make therapeutic proteins, most of them employing CHO or E. coli cell-free extracts. Here, we propose an alternative that uses human blood derived leukocyte cell extracts for the expression of recombinant proteins. We demonstrate expression of nano luciferase (Nluc), Granulocyte-colony stimulating factor (G-CSF) and Erythropoietin (EPO) in cell-free leukocyte extracts within two hours. Human blood is readily available from donors and blood banks and leukocyte rich fractions are easy to obtain. The method described here demonstrates the ability to rapidly express recombinant proteins from human cell extracts that could provide the research community with a facile technology to make their target protein. Eventually, we envision that any recombinant protein can be produced from patient-supplied leukocytes, which can then be injected back into the patient. This approach could lead to an alternative model for personalized medicines and vaccines.
Vibrio natriegens constitutes one of the fastest-growing nonpathogenic bacteria and a potential novel workhorse for many biotechnological applications. Here, we report the development of a Vibrio-based cell-free protein synthesis system (CFPS). Specifically, up to 0.4 g L⁻¹ eGFP could be successfully synthesized in small-scale batch reactions using cell-free extract obtained from fast-growing V. natriegens cultures. Versatile CFPS system characterization attained by combining the analyses of key metabolites for translation and ribosomes revealed limitations regarding rRNA stability and critical substrate consumption (e.g., amino acids). Alternatively, rRNA showed increased stability by inducing Mg²⁺homeostasis in the reaction. Although the enormous translation capacity of the CFPS system based on the available ribosome concentration could not yet be fully exploited, its potential was successfully demonstrated by activating an endogenous transcription unit with V. natriegensRNA polymerase (RNAP) for protein expression. This allowed the use of in vitro screening for promoter strength, a critical factor for efficient gene expression in vitro and in vivo. Three different promoters were tested and output signals corresponded well with the expected affinity for V. natriegens RNAP. This established CFPS toolbox may provide a foundation to establish V. natriegens as a valuable platform in biotechnology as well as synthetic biology.
The fast growing bacterium Vibrio natriegens is an emerging microbial host for biotechnology. Harnessing its productive cellular components may offer a compelling platform for rapid protein production and prototyping of metabolic pathways or genetic circuits. Here, we report the development of a V. natriegens cell-free expression system. We devised a simplified crude extract preparation protocol and achieved >260μg/mL of super-folder GFP in a small-scale batch reaction after three hours. Culturing conditions, including growth media and cell density, significantly affect translation kinetics and protein yield of extracts. We observed maximal protein yield at incubation temperatures of 26°C or 30°C, and show improved yield by tuning ions crucial for ribosomal stability. This work establishes an initial V. natriegens cell-free expression system, enables probing of V. natriegens biology, and will serve as a platform to accelerate metabolic engineering and synthetic biology applications.
Cell-free protein synthesis (CFPS) systems enable the production of protein without the use of living, intact cells. An emerging area of interest is to use CFPS systems to characterize individual elements for genetic programs [e.g. promoters, ribosome binding sites (RBS)]. To enable this research area, robust CFPS systems must be developed from new chassis organisms. One such chassis is the Gram-negative Pseudomonas bacteria, which have been studied extensively for their diverse metabolism with promises in the field of bioremediation and biosynthesis. Here, we report the development and optimization of a high-yielding (198 ± 5.9 µg/ml) batch CFPS system from Pseudomonas putida ATCC 12633. Importantly, both circular and linear DNA templates can be applied directly to the CFPS reaction to program protein synthesis. Therefore, it is possible to prepare hundreds or even thousands of DNA templates without time-consuming cloning work. This opens the possibility to rapidly assess and validate genetic part performance in vitro before performing experiments in cells. To validate the P. putida CFPS system as a platform for prototyping genetic parts, we designed and constructed a library consisting of 15 different RBSs upstream of the reporter protein sfGFP, which covered an order of magnitude range in expression. Looking forward, our P. putida CFPS platform will not only expand the protein synthesis toolkit for synthetic biology but also serve as a platform in expediting the screening and prototyping of gene regulatory elements.
Despite its early promise as a diagnostic and prognostic tool, gene expression profiling remains cost-prohibitive and challenging to implement in a clinical setting. Here, we introduce a molecular computation strategy for analysing the information contained in complex gene expression signatures without the need for costly instrumentation. Our workflow begins by training a computational classifier on labelled gene expression data. This in silico classifier is then realized at the molecular level to enable expression analysis and classification of previously uncharacterized samples. Classification occurs through a series of molecular interactions between RNA inputs and engineered DNA probes designed to differentially weigh each input according to its importance. We validate our technology with two applications: a classifier for early cancer diagnostics and a classifier for differentiating viral and bacterial respiratory infections based on host gene expression. Together, our results demonstrate a general and modular framework for low-cost gene expression analysis.
Taking CRISPR technology further
CRISPR techniques are allowing the development of technologies for nucleic acid detection (see the Perspective by Chertow). Taking advantages of the distinctive enzymatic properties of CRISPR enzymes, Gootenberg et al. developed an improved nucleic acid detection technology for multiplexed quantitative and highly sensitive detection, combined with lateral flow for visual readout. Myhrvold et al. added a sample preparation protocol to create a field-deployable viral diagnostic platform for rapid detection of specific strains of pathogens in clinical samples. Cas12a (also known as Cpf1), a type V CRISPR protein, cleaves double-stranded DNA and has been adapted for genome editing. Chen et al. discovered that Cas12a also processes single-stranded DNA threading activity. A technology platform based on this activity detected human papillomavirus in patient samples with high sensitivity.
Science , this issue p. 439 , p. 444 , p. 436 ; see also p. 381
Significance
Nonmodel bacteria have essential roles to play in the future development of biotechnology by providing new sources of biocatalysts, antibiotics, hosts for bioproduction, and engineered “living therapies.” The characterization of such hosts can be challenging, as many are not tractable to standard molecular biology techniques. This paper presents a rapid and automated methodology for characterizing new DNA parts from a nonmodel bacterium using cell-free transcription–translation. Data analysis was performed with Bayesian parameter inference to provide an understanding of gene-expression dynamics and resource sharing. We suggest that our integrated approach is expandable to a whole range of nonmodel bacteria for the characterization of new DNA parts within a native cell-free background for new biotechnology applications.
The most common method for quantifying small-molecule drugs in blood samples is by liquid chromatography in combination with mass spectrometry. Few immuno-based assays are available for the detection of small-molecule drugs in blood. Here we report on a homogeneous assay that enables detection of the concentration of digoxin spiked into in a plasma sample. The assay is based on a shift in the equilibrium of a DNA strand displacement competition reaction, and can be performed in 30 min for concentrations above 10 nM. The equilibrium shift occurs upon binding of anti-digoxigenin antibody. As a model, the assay provides a potential alternative to current small-molecule detection methods used for therapeutic drug monitoring.
Many diseases and disorders are linked to exposure to endocrine disrupting chemicals (EDCs) that mimic the function of natural estrogen hormones. Here we present a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting chemicals that interact with the human estrogen receptor β (hERβ). This biosensor consists of an allosteric fusion protein, which is expressed using cell-free protein synthesis technology and is directly assayed by a colorimetric response. The resultant biosensor successfully detected known EDCs of hERβ (BPA, E2, and DPN) at similar or better detection range than an analogous cell-based biosensor, but in a fraction of time. We also engineered cell-free protein synthesis reactions with RNAse inhibitors to increase production yields in the presence of human blood and urine. The RAPID biosensor successfully detects EDCs in these human samples in the presence of RNAse inhibitors. Engineered cell-free protein synthesis facilitates the use of protein biosensors in complex sample matrices without cumbersome protein purification.
Taking CRISPR technology further
CRISPR techniques are allowing the development of technologies for nucleic acid detection (see the Perspective by Chertow). Taking advantages of the distinctive enzymatic properties of CRISPR enzymes, Gootenberg et al. developed an improved nucleic acid detection technology for multiplexed quantitative and highly sensitive detection, combined with lateral flow for visual readout. Myhrvold et al. added a sample preparation protocol to create a field-deployable viral diagnostic platform for rapid detection of specific strains of pathogens in clinical samples. Cas12a (also known as Cpf1), a type V CRISPR protein, cleaves double-stranded DNA and has been adapted for genome editing. Chen et al. discovered that Cas12a also processes single-stranded DNA threading activity. A technology platform based on this activity detected human papillomavirus in patient samples with high sensitivity.
Science , this issue p. 439 , p. 444 , p. 436 ; see also p. 381
Assembly of recombinant multiprotein systems requires multiple culturing and purification steps that scale linearly with the number of constituent proteins. This problem is particularly pronounced in the preparation of the 34 proteins involved in transcription and translation systems, which are fundamental biochemistry tools for reconstitution of cellular pathways ex vivo. Here, we engineer synthetic microbial consortia consisting of between 15 and 34 Escherichia coli strains to assemble the 34 proteins in a single culturing, lysis, and purification procedure. The expression of these proteins is controlled by synthetic genetic modules to produce the proteins at the correct ratios. We show that the pure multiprotein system is functional and reproducible, and has low protein contaminants. We also demonstrate its application in the screening of synthetic promoters and protease inhibitors. Our work establishes a novel strategy for producing pure translation machinery, which may be extended to the production of other multiprotein systems.
A longstanding goal of synthetic biology has been the programmable control of cellular functions. Central to this is the creation of versatile regulatory toolsets that allow for programmable control of gene expression. Of the many regulatory molecules available, RNA regulators offer the intriguing possibility of de novo design—allowing for the bottom-up molecular-level design of genetic control systems. Here we present a computational design approach for the creation of a bacterial regulator called Small Transcription Activating RNAs (STARs) and create a library of high-performing and orthogonal STARs that achieve up to ~ 9000-fold gene activation. We demonstrate the versatility of these STARs—from acting synergistically with existing constitutive and inducible regulators, to reprogramming cellular phenotypes and controlling multigene metabolic pathway expression. Finally, we combine these new STARs with themselves and CRISPRi transcriptional repressors to deliver new types of RNA-based genetic circuitry that allow for sophisticated and temporal control of gene expression.
The common smut of corn, caused by Ustilago maydis is a troublesome disease of maize. Early and accurate detection of U. maydis is essential for the disease management. In this study, primer set Pep-2 was selected for LAMP (loop-mediated isothermal amplification) from 12 sets of primers targeting three U. maydis effector genes See1, Pit2 and Pep1 according to primer screening. The optimal concentrations of Bst DNA polymerase and Mg²⁺ as well as inner/outer primer ratio of the LAMP reaction system were screened by combining a single factor experiment and an orthogonal design arrangement. The specificity of this real-time LAMP (RealAmp) assay was confirmed by negative testing for other pathogens. The detection sensitivity of the RealAmp assay was 200 times higher than that of detection through conventional PCR. Results of the RealAmp assay for quantifying the genomic DNA of U. maydis were confirmed by testing with both artificially and naturally infected samples. In addition, the RealAmp reaction could be conducted via an improved tube scanner to implement a “electricity free” assay from template preparation to quantitative detection. The resulting assay could be more convenient for use in the field as a simple, rapid, and effective technique for monitoring U. maydis.
Coxiella burnetii, the causative pathogen for Q fever, is an obligate intracellular bacterium and designated as a biosafety level 3 agent. Detection and quantification of the bacteria with conventional culturing methods is time-consuming and poses significant health risks. Polymerase chain reaction (PCR)-based assays have been developed for detecting C. burnetii and could provide rapid diagnosis. However, they require specialized equipment, including a cold chain for PCR reagents that maintains their stability during storage and transport. These requirements limit the advantage of PCR-based methods, especially in resource-limited areas.
Previously, we had developed a lyophilized loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii. To simplify and improve this assay, the reagents for the LAMP assay and the detecting reagent, SYBR green, were lyophilized together. The stability of the lyophilized reagents was evaluated by measuring changes in detection limit for plasmid DNA encoding a C. burnetii gene upon storage at 4 °C, 25 °C, or 37 °C. Our data indicate that the lyophilized reagents remain stable for 24 months when stored at 4 °C, 28 days at 25 °C, and 2 days at 37 °C. This improved LAMP assay can be easily performed in a simple water bath or heating block. The stability at ambient temperature, the simplicity of assay procedure, and the availability of low cost equipment make this method ideal for use in resource-limited settings where Q fever is endemic.
Synthetic biology designed cell-free biosensors are a promising new tool for the detection of clinically relevant biomarkers in infectious diseases. Here, we report that a modular DNA-encoded biosensor in cell-free protein expression systems can be used to measure a bacterial biomarker of Pseudomonas aeruginosa infection from human sputum samples. By optimizing the cell-free system and sample extraction, we demonstrate that the quorum sensing molecule 3-oxo-C12-HSL in sputum samples from cystic fibrosis lungs can be quantitatively measured at nanomolar levels using our cell-free biosensor system, and is comparable to LC–MS measurements of the same samples. This study further illustrates the potential of modular cell-free biosensors as rapid, low-cost detection assays that can inform clinical practice.
Quantitative measurement of transcription rates in live cells is important for revealing mechanisms of transcriptional regulation. This is particularly challenging when measuring the activity of RNA polymerase III (Pol III), which transcribes growth-promoting small RNAs. To address this issue, we developed Corn, a genetically encoded fluorescent RNA reporter suitable for quantifying RNA transcription in cells. Corn binds and induces fluorescence of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime, which resembles the fluorophore found in red fluorescent protein (RFP). Notably, Corn shows high photostability, enabling quantitative fluorescence imaging of mTOR-dependent Pol III transcription. We found that, unlike actinomycin D, mTOR inhibitors resulted in heterogeneous transcription suppression in individual cells. Quantitative imaging of Corn-tagged Pol III transcript levels revealed distinct Pol III transcription 'trajectories' elicited by mTOR inhibition. Together, these studies provide an approach for quantitative measurement of Pol III transcription by direct imaging of Pol III transcripts containing a photostable RNA–fluorophore complex.
The use of cell-free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell-free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFPi and streptokinase in a cell-free system using instrumented mini-bioreactors for highly reproducible protein production. We achieved recombinant protein production (∼600 µg/mL of tGFP and 500 µg/mL streptokinase in 2.5 h of expression time, comparable to previously reported yields for cell-free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare these to a tag-free self-cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose-level production of therapeutic proteins at the point-of-care. This article is protected by copyright. All rights reserved
Cell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biology and biotechnology applications, including production of robust field-ready biosensors. Here, we combine programmed cellular autolysis with a freeze-thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid production of bacterial lysates for cell-free gene expression. Using this method, robust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible. Moreover, live autolysis strain can be freeze-dried directly and subsequently lysed upon rehydration to produce active lysate. We demonstrate the utility of autolysates for synthetic biology by regulating protein production and degradation, implementing quorum sensing, and showing quantitative protection of linear DNA templates by GamS protein. To allow versatile and sensitive beta-galactosidase (LacZ) based readout we produce autolysates with no detectable background LacZ activity and use them to produce sensitive mercury(II) biosensors with LacZ-mediated colorimetric and fluorescent outputs. The autolysis approach can facilitate wider adoption of cell-free technology for cell-free gene expression as well as other synthetic biology and biotechnology applications, such as metabolic engineering, natural product biosynthesis, or proteomics.
Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play a variety of important regulatory roles in many eukaryotes. Their small size has made it challenging to study them directly in live cells. Here we describe an RNA-based fluorescent sensor for small RNA detection both in vitro and in vivo, adaptable for any small RNA. It utilizes an sxRNA switch for detection of miRNA-mRNA interactions combined with a fluorophore-binding sequence 'Spinach', a GFP-like RNA aptamer for which the RNA-fluorophore complex exhibits strong and consistent fluorescence under an excitation wavelength. Two example sensors, FASTmiR171 and FASTmiR122, can rapidly detect and quantify the levels of miR171 and miR122 in vitro. The sensors can determine relative levels of miRNAs in total RNA extracts with sensitivity similar to small RNA sequencing and northern blots. FASTmiR sensors were also used to estimate the copy number range of miRNAs in total RNA extracts. To localize and analyze the spatial distribution of small RNAs in live, single cells, tandem copies of FASTmiR122 were expressed in different cell lines. FASTmiR122 was able to quantitatively detect the differences in miR122 levels in Huh7 and HEK293T cells demonstrating its potential for tracking miRNA expression and localization in vivo.
Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test
platforms for point-of-care (POC) testing because they have simplicity, portability, and
cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT),
the development of NAT based on a paper platform has not progressed as much as the others
because various specific conditions for nucleic acid amplification reactions such as pH, buffer
components, and temperature, inhibitions from technical differences of paper-based device. Here,
we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP)
with real-time simultaneous detection of multiple DNA targets. We determined the optimal
chemical components to enable dry conditions for the LAMP reaction without lyophilization or
other techniques. We also devised the simple paper device structure by sequentially stacking
functional layers, and employed a newly discovered property of hydroxynaphthol blue
fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform
allowed analysis of three different meningitis DNA samples in a single device with single-step
operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with
quantitative detection of 102
–105 copies of genomic DNA. Furthermore, we propose the
transformation of DNA amplification devices to a simple and affordable paper system approach
with great potential for realizing a paper-based NAT system for POC testing.
Bacteria can be engineered to function as diagnostics or therapeutics in the mammalian gut but commercial translation of technologies to accomplish this has been hindered by the susceptibility of synthetic genetic circuits to mutation and unpredictable function during extended gut colonization. Here, we report stable, engineered bacterial strains that maintain their function for 6 months in the mouse gut. We engineered a commensal murine Escherichia coli strain to detect tetrathionate, which is produced during inflammation. Using our engineered diagnostic strain, which retains memory of exposure in the gut for analysis by fecal testing, we detected tetrathionate in both infection-induced and genetic mouse models of inflammation over 6 months. The synthetic genetic circuits in the engineered strain were genetically stable and functioned as intended over time. The durable performance of these strains confirms the potential of engineered bacteria as living diagnostics.
RNA-RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA-RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In contrast, RNA aptamers that sequester small molecule dyes and activate their fluorescence are increasingly utilized in genetically encoded strategies to report on RNA-level events. Split-aptamer systems have been rationally designed to generate signal upon hybridization of two or more discrete RNA transcripts, but none directly function when expressed in vivo. We reasoned that the improved physiological properties of the Broccoli aptamer enable construction of a split-aptamer system that could function in living cells. Here we present the Split-Broccoli system, in which self-assembly is nucleated by a thermostable, three-way junction RNA architecture and fluorescence activation requires both strands. Functional assembly of the system approximately follows second-order kinetics in vitro and improves when cotranscribed, rather than when assembled from purified components. Split-Broccoli fluorescence is digital in vivo and retains functional modularity when fused to RNAs that regulate circuit function through RNA-RNA hybridization, as demonstrated with an RNA Toehold switch. Split-Broccoli represents the first functional split-aptamer system to operate in vivo. It offers a genetically encoded and nondestructive platform to monitor and exploit RNA-RNA hybridization, whether as an all-RNA, stand-alone AND gate or as a tool for monitoring assembly of RNA-RNA hybrids.
Many biotechnology capabilities are limited by stringent storage needs of reagents, largely prohibiting use outside of specialized laboratories. Focusing on a large class of protein-based biotechnology applications, we address this issue by developing a method for preserving cell-free protein expression systems for months above room temperature. Our approach realizes unprecedented long-term stability at elevated temperatures by leveraging the sugar alcohol trehalose, a simple, low-cost, open-air drying step, and strategic separation of reaction components during drying. The resulting preservation capacity enables efficient production of a wide range of on-demand proteins under adverse conditions, for instance during emergency outbreaks or in remote locations. To demonstrate application potential, we use cell-free reagents subjected to months of exposure at 37°C and atmospheric conditions to produce sufficient concentrations of a pyocin protein to kill Pseudomonas aeruginosa, a troublesome pathogen for traumatic and burn wound injuries. Our work makes possible new biotechnology applications that demand ruggedness and scalability.
Sensitive and specific CRISPR diagnostics
Methods are needed that can easily detect nucleic acids that signal the presence of pathogens, even at very low levels. Gootenberg et al. combined the allele-specific sensing ability of CRISPR-Cas13a with recombinase polymerase amplification methods to detect specific RNA and DNA sequences. The method successfully detected attomolar levels of Zika virus, as well as the presence of pathogenic bacteria. It could also be used to perform human genotyping from cell-free DNA.
Science , this issue p. 438
Here we introduce a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting ligands that interact with NHRs. The RAPID platform can be adapted for field use, allowing rapid evaluation of EDC presence or absence in environmental samples, and could also be applied for drug screening. The biosensor is based on an engineered, allosterically-activated fusion protein, which contains the ligand-binding domain (LBD) from a target NHR (human thyroid receptor in this work). In vitro expression of this protein using cell-free protein synthesis (CFPS) technology in the presence of an EDC leads to activation of a reporter enzyme, reported through a straightforward colorimetric assay output. In this work, we demonstrate the potential of this biosensor platform to be used in a portable “just-add-sample” format for near real-time detection. We also demonstrate the robust nature of the cell-free protein synthesis component in the presence of a variety of environmental and human samples, including sewage, blood, and urine. The presented RAPID biosensor platform is significantly faster and less labor intensive than commonly available technologies, making it a promising tool for detecting environmental EDC contamination and screening potential NHR-targeted pharmaceuticals.
Background
Current diagnostic methods for Schistosoma japonicum infection are insensitive for low-density infections. Therefore, a new diagnostic assay based on recombinase polymerase amplification (RPA) technology was established and assessed for field applification. Methods
The S.japonicum RPA assay was developed to target highly repetitive retrotransposon SjR2 gene of S japonicum, and its sensitivity and specificity were assessed by serial dilution of S. japonicum genomic DNA and other related worm genomic DNA respectively. The RPA diagnostic validity was first evaluated in 60 fecal samples from healthy people and patients, and then compared with other diagnostic tests in 200 high-risk individuals living in endemic areas. ResultsThe real time RPA assay could detect 0.9 fg S. japonicum DNA within 15 min and distinguish S. japonicum from other worms. The validity analysis of RPA for the detection of S. japonicum in stool samples from 30 S. japonicum-infected patients and 30 healthy persons indicated 100% sensitivity and specificity. When testing 200 fecal or serum samples from a high-risk population, the percentage sensitivity of RPA was 100%, whereas that of indirect hemagglutination assay (IHA) and enzyme-linked immunosorbent assay (ELISA) were 80.3% and 85.2% respectively. In addition, the RPA presented better consistency with the stool-based tests than IHA and ELISA. Overall, the RPA was superior to other detection methods with respect to detection time, sensitivity, and convenience. Conclusions
This is the first time we applied the RPA technology to the field evaluation of S. japonicum infection. And the results suggest that RPA-based assays can be used as a promising point-of-care test for the diagnosis of schistosomiasis.
The fields of biosensing and bioremediation leverage the phenomenal array of sensing and metabolic capabilities offered by natural microbes. Synthetic biology provides tools for transforming these fields through complex integration of natural and novel biological components to achieve sophisticated sensing, regulation, and metabolic function. However, the majority of synthetic biology efforts are conducted in living cells, and concerns over releasing genetically modified organisms constitute a key barrier to environmental applications. Cell-free protein expression systems offer a path towards leveraging synthetic biology, while preventing the spread of engineered organisms in nature. Recent efforts in the areas of cell-free approaches for sensing, regulation, and metabolic pathway implementation, as well as for preserving and deploying cell-free expression components, embody key steps towards realizing the potential of cell-free systems for environmental sensing and remediation.
DNA-based molecular circuits allow autonomous signal processing, but their actuation has relied mostly on RNA/DNA-based inputs, limiting their application in synthetic biology, biomedicine and molecular diagnostics. Here we introduce a generic method to translate the presence of an antibody into a unique DNA strand, enabling the use of antibodies as specific inputs for DNA-based molecular computing. Our approach, antibody-templated strand exchange (ATSE), uses the characteristic bivalent architecture of antibodies to promote DNA-strand exchange reactions both thermodynamically and kinetically. Detailed characterization of the ATSE reaction allowed the establishment of a comprehensive model that describes the kinetics and thermodynamics of ATSE as a function of toehold length, antibody–epitope affinity and concentration. ATSE enables the introduction of complex signal processing in antibody-based diagnostics, as demonstrated here by constructing molecular circuits for multiplex antibody detection, integration of multiple antibody inputs using logic gates and actuation of enzymes and DNAzymes for signal amplification.
Cell-free protein synthesis (CFPS) has emerged as a powerful platform for applied biotechnology and synthetic biology, with a range of applications in synthesizing proteins, evolving proteins, and prototyping genetic circuits. To expand the current CFPS repertoire, we report here the development and optimization of a Streptomyces-based CFPS system for the expression of GC-rich genes. By developing a streamlined crude extract preparation protocol and optimizing reaction conditions, we were able to achieve active enhanced green fluorescent protein (EGFP) yields of greater than 50 µg/mL with batch reactions lasting up to 3 h. By adopting a semi-continuous reaction format, the EGFP yield could be increased to 282 ± 8 µg/mL and the reaction time was extended to 48 h. Notably, our extract preparation procedures were robust to multiple Streptomyces lividans and Streptomyces coelicolor strains, although expression yields varied. We show that our optimized Streptomyces lividans system provides benefits when compared to an Escherichia coli-based CFPS system for increasing percent soluble protein expression for four Streptomyces-originated high GC-content genes that are involved in biosynthesis of the nonribosomal peptides tambromycin and valinomycin. Looking forward, we believe that our Streptomyces-based CFPS system will contribute significantly towards efforts to express complex natural product gene clusters (e.g., nonribosomal peptides and polyketides), providing a new avenue for obtaining and studying natural product biosynthesis pathways. This article is protected by copyright. All rights reserved
Human norovirus is a leading cause of viral gastroenteritis worldwide. Rapid detection could facilitate control, however widespread point-of-care testing is infrequently done due to the lack of robust and portable methods. Recombinase polymerase amplification (RPA) is a novel isothermal method which rapidly amplifies and detects nucleic acids using a simple device in near real-time. An RT-RPA assay targeting a recent epidemic human norovirus strain (GII.4 New Orleans) was developed and evaluated in this study. The assay successfully detected purified norovirus RNA from multiple patient outbreak isolates and had a limit of detection of 3.40 ± 0.20 log10 genomic copies (LGC), which is comparable to most other reported isothermal norovirus amplification methods. The assay also detected norovirus in directly boiled stool, and displayed better resistance to inhibitors than a commonly used RT-qPCR assay. The assay was specific, as it did not amplify genomes from 9 non-related enteric viruses and bacteria. The assay detected norovirus in some samples in as little as 6 min, and the entire detection process can be performed in less than 30 min. The reported RT-RPA method shows promise for sensitive point-of-care detection of epidemic human norovirus, and is the fastest human norovirus amplification method to date.
Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8 µM GFP, which gave a ~72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type Pgrac (σA) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase.
Chemical sensors are self-contained analytical devices that provide real-time information on chemical composition. A chemical sensor integrates two distinct functions: recognition and transduction. Such devices are widely used for a broad variety of applications, including clinical analysis, environment monitoring and monitoring of industrial processes. This text provides an up-to-date survey of chemical sensor science and technology, with a good balance between classical aspects and contemporary trends. Nanomaterials applications are amply presented in two dedicated chapters (8 and 20) as well in various sections throughout other chapters.
Suitable as a textbook for graduate and final year undergraduate students, and also for researchers in chemistry, biology, physics, physiology, pharmacology and electronic engineering, this book is valuable to anyone interested in the field of chemical sensors and biosensors.
Optogenetic tools provide a new and efficient way to dynamically program gene expression with unmatched spatiotemporal precision. To date, its vast potential remains untapped in the field of cell-free synthetic biology, largely due to the lack of simple and efficient light-switchable systems. Here, to bridge the gap between cell-free systems and optogenetics, we studied our previously engineered one component-based blue light-inducible Escherichia coli promoter in a cell-free environment through experimental characterization and mathematical modelling. We achieved >10-fold dynamic expression and demonstrated rapid and reversible activation of target gene to generate oscillatory waveform. Deterministic model developed was able to recapitulate the system behaviour and helped to provide quantitative insights to optimize dynamic response. This in vitro optogenetic approach could be a powerful new high-throughput screening technology for rapid prototyping of complex biological networks in both space and time without the need for chemical induction.
The genotyping of a single-nucleotide polymorphism (SNP) is addressed through methods based on loop-mediated isothermal amplification (LAMP) combined with user-friendly optical read-outs to cover the current demand for point-of-care DNA biomarker detection. The modification of primer design and reaction composition improved the assay selectivity yielding allele-specific results and reducing false-positive frequency. Furthermore, the reduced cost, ease of use and effectiveness of colorimetric detection (solution and hybridisation chip formats) were availed for the image capture by a smartphone, reching high sensitivity. In order to evaluate their discriminating capacities, LAMP-based methods were applied to human samples to genotype a SNP biomarker (rs1954787) located in the GRIK4 gene and related to the treatment response to anti-depressants drugs. Sensitive (limit of detection: 100 genomic DNA copies), reproducible (< 15% error), fast (around 70 min) and low-cost assays were accomplished. Patient subgroups were correctly discriminated, agreeing with reference sequencing techniques. The achieved analytical performances using the developed amplification-detection principles confirmed the approach potential for point-of-care optical DNA testing.
Taking CRISPR technology further
CRISPR techniques are allowing the development of technologies for nucleic acid detection (see the Perspective by Chertow). Taking advantages of the distinctive enzymatic properties of CRISPR enzymes, Gootenberg et al. developed an improved nucleic acid detection technology for multiplexed quantitative and highly sensitive detection, combined with lateral flow for visual readout. Myhrvold et al. added a sample preparation protocol to create a field-deployable viral diagnostic platform for rapid detection of specific strains of pathogens in clinical samples. Cas12a (also known as Cpf1), a type V CRISPR protein, cleaves double-stranded DNA and has been adapted for genome editing. Chen et al. discovered that Cas12a also processes single-stranded DNA threading activity. A technology platform based on this activity detected human papillomavirus in patient samples with high sensitivity.
Science , this issue p. 439 , p. 444 , p. 436 ; see also p. 381
Antibody detection plays a pivotal role in the diagnosis of pathogens and monitoring the success of vaccine immunization. However, current serology techniques require multiple, time-consuming washing and incubation steps, which limit their applicability in point-of-care (POC) diagnostics and high-throughput assays. We developed here a nucleic acid nanoswitch platform able to instantaneously measure immunoglobulins of type G and E (IgG and IgE) levels directly in blood serum and other bodily fluids. The system couples the advantages of target-binding induced colocalization and nucleic acid conformational-change nanoswitches. Due to the modular nature of the recognition platform, the method can potentially be applied to the detection of any antibody for which an antigen can be conjugated to a nucleic acid strand. In this work we show the sensitive, fast and cost-effective detection of four different antibodies and demonstrate the possible use of this approach for the monitoring of antibody levels in HIV+ patients immunized with AT20 therapeutic vaccine.
Cell-free protein synthesis (CFPS) has emerged as a powerful approach to recombinant protein biosynthesis for applications in biochemical engineering and synthetic biology. To date, CFPS systems have been most commonly derived from a variety of organism sources including microbes (e.g., Escherichia coli and yeast), plants (e.g., wheat germ and tobacco), and eukaryotic cells (e.g., rabbit reticulocytes and Chinese Hamster Ovary cells), each with their own advantages and opportunities. To expand the palette of CFPS platforms, we recently established a Streptomyces lividans-based cell-free system for the expression, especially, of high GC-content genes that are involved in the biosynthesis of natural products. Unfortunately, batch protein expression yields were limited to ∼50 μg/mL of a model enhanced green fluorescent protein (EGFP). Here, we sought to address this limitation and improve protein biosynthesis yields. By increasing the total extract protein concentration in the CFPS reaction, which increases the concentration of catalyst proteins available for protein biosynthesis and energy regeneration, and modifying our extract preparation procedure, we enhanced batch protein biosynthesis yields of EGFP more than 2-fold, to 116.9 ± 8.2 μg/mL. Then, we demonstrated that our simple and robust approach could be applied to six other Streptomyces strains. Looking forward, we expect that our more highly productive and efficient Streptomyces CFPS systems can be used to synthesize, study, and discover natural product biosynthesis pathways in vitro.
Recombinase polymerase amplification (RPA) is a highly sensitive and selective isothermal amplification technique, operating at 37–42°C, with minimal sample preparation and capable of amplifying as low as 1–10 DNA target copies in less than 20 min. It has been used to amplify diverse targets, including RNA, miRNA, ssDNA and dsDNA from a wide variety of organisms and samples. An ever increasing number of publications detailing the use of RPA are appearing and amplification has been carried out in solution phase, solid phase as well as in a bridge amplification format. Furthermore, RPA has been successfully integrated with different detection strategies, from end-point lateral flow strips to real-time fluorescent detection amongst others. This review focuses on the different methodologies and advances related to RPA technology, as well as highlighting some of the advantages and drawbacks of the technique.
Synthetic biology aims to develop engineering-driven approaches to the programming of cellular functions that could yield transformative technologies. Synthetic gene circuits that combine DNA, protein, and RNA components have demonstrated a range of functions such as bistability, oscillation, feedback, and logic capabilities. However, it remains challenging to scale up these circuits owing to the limited number of designable, orthogonal, high-performance parts, the empirical and often tedious composition rules, and the requirements for substantial resources for encoding and operation. Here, we report a strategy for constructing RNA-only nanodevices to evaluate complex logic in living cells. Our 'ribocomputing' systems are composed of de-novo-designed parts and operate through predictable and designable base-pairing rules, allowing the effective in silico design of computing devices with prescribed configurations and functions in complex cellular environments. These devices operate at the post-transcriptional level and use an extended RNA transcript to co-localize all circuit sensing, computation, signal transduction, and output elements in the same self-assembled molecular complex, which reduces diffusion-mediated signal losses, lowers metabolic cost, and improves circuit reliability. We demonstrate that ribocomputing devices in Escherichia coli can evaluate two-input logic with a dynamic range up to 900-fold and scale them to four-input AND, six-input OR, and a complex 12-input expression (A1 AND A2 AND NOT A1*) OR (B1 AND B2 AND NOT B2*) OR (C1 AND C2) OR (D1 AND D2) OR (E1 AND E2). Successful operation of ribocomputing devices based on programmable RNA interactions suggests that systems employing the same design principles could be implemented in other host organisms or in extracellular settings.
Light-up RNA aptamers are valuable tools for fluorescence imaging of RNA in living cells and thus for elucidating RNA functions and dynamics. However, no light-up RNA sensor has been reported for imaging of microRNAs (miRs) in mammalian cells. We herein report a novel genetically-encoded RNA sensor for fluorescent imaging of miRs in liv-ing tumor cells using a light-up RNA aptamer which binds to sulforhodamine and separates it from a conjugated contact quencher. Based on the structural switching mechanism characteristic for molecular beacon, we show that the RNA sensor activates high-contrast fluorescence from the sulfor-hodamine-quencher conjugate when its stem-loop respon-sive motif hybridizes with target miR. The RNA sensor can be stably expressed within a designed tRNA scaffold in tumor cells and deliver light-up response to target miR. We also realize the RNA sensor for dual-emission, ratiometric imag-ing by coexpression of RNA sensor and GFP, enabling quanti-tative studies of target miR in living cells. Our design may provide a new paradigm for developing robust, sensitive light-up RNA sensors for RNA imaging applications.
Complex DNA sequences are difficult to detect and profile, but are important contributors to human health and disease. Existing hybridization probes lack the capability to selectively bind and enrich hypervariable, long or repetitive sequences. Here, we present a generalized strategy for constructing modular hybridization probes (M-Probes) that overcomes these challenges. We demonstrate that M-Probes can tolerate sequence variations of up to 7 nt at prescribed positions while maintaining single nucleotide sensitivity at other positions. M-Probes are also shown to be capable of sequence-selectively binding a continuous DNA sequence of more than 500 nt. Furthermore, we show that M-Probes can detect genes with triplet repeats exceeding a programmed threshold. As a demonstration of this technology, we have developed a hybrid capture method to determine the exact triplet repeat expansion number in the Huntington's gene of genomic DNA using quantitative PCR.
A distance-based paper analytical device (dPAD) for loop mediated isothermal amplification (LAMP) detection based on distance measurement was proposed. This approach relied on visual detection by the length of colour developed on the dPAD with reference to semi-quantitative determination of the initial amount of genomic DNA. In this communication, E. coli DNA was chosen as a template DNA for LAMP reaction. In accordance with the principle, the dPAD was immobilized by polyethylenimine (PEI), which is a strong cationic polymer, in the hydrophilic channel of the paper device. Hydroxynaphthol blue (HNB), a colourimetric indicator for monitoring the change of magnesium ion concentration in the LAMP reaction, was used to react with the immobilized PEI. The positive charges of PEI react with the negative charges of free HNB in the LAMP reaction, producing a blue colour deposit on the paper device. Consequently, the apparently visual distance appeared within 5 minutes and length of distance correlated to the amount of DNA in the sample. The distance-based PAD for the visual detection of the LAMP reaction could quantify the initial concentration of genomic DNA as low as 4.14×10³ copies µL⁻¹. This distance-based visual semi-quantitative platform is suitable for choice of LAMP detection method, particular in resource-limited settings because of the advantages of low cost, simple fabrication and operation, disposability and portable detection of the dPAD device.
Metal ions are essential to many chemical, biological, and environmental processes. In the past two decades, many DNA-based metal sensors have emerged. While the main biological role of DNA is to store genetic information, its chemical structure is ideal for metal binding via both the phosphate backbone and nucleobases. DNA is highly stable, cost-effective, easy to modify, and amenable to combinatorial selection. Two main classes of functional DNA were developed for metal sensing: aptamers and DNAzymes. While a few metal binding aptamers are known, it is generally quite difficult to isolate such aptamers. On the other hand, DNAzymes are powerful tools for metal sensing since they are selected based on catalytic activity, thus bypassing the need for metal immobilization. In the last five years, a new surge of development has been made on isolating new metal-sensing DNA sequences. To date, many important metals can be selectively detected by DNA often down to the low parts-per-billion level. Herein, each metal ion and the known DNA sequences for its sensing are reviewed. We focus on the fundamental aspect of metal binding, emphasizing the distinct chemical property of each metal. Instead of reviewing each published sensor, a high-level summary of signaling methods is made as a separate section. In principle, each signaling strategy can be applied to many DNA sequences for designing sensors. Finally, a few specific applications are highlighted, and future research opportunities are discussed.
Optogenetic tools use colored light to rapidly control gene expression in space and time. We designed a genetically encoded system that gives Escherichia coli the ability to distinguish between red, green, and blue (RGB) light and respond by changing gene expression. We use this system to produce 'color photographs' on bacterial culture plates by controlling pigment production and to redirect metabolic flux by expressing CRISPRi guide RNAs. © 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
Chinese Hamster Ovary (CHO) cells are routinely optimized to stably express monoclonal antibodies (mAbs) at high titers. At the early stages of lead isolation and optimization, hundreds of sequences for the target protein of interest are screened. Typically, cell-based transient expression technology platforms are used for expression screening, but these can be time- and resource-intensive. Here, we have developed a cell-free protein synthesis (CFPS) platform utilizing a commercially available CHO extract for the rapid in vitro synthesis of active, aglycosylated mAbs. Specifically, we optimized reaction conditions to maximize protein yields, established an oxidizing environment to enable disulfide bond formation, and demonstrated the importance of temporal addition of heavy chain and light chain plasmids for intact mAb production. Using our optimized platform, we demonstrate for the first time to our knowledge the CFPS of biologically active, intact mAb at >100 mg/L using a eukaryotic-based extract. We then also explored the utility of our system as a tool for ranking yields of candidate antibodies. Unlike stable or transient transfection-based screening, which requires a minimum of 7 days for setup and execution, results using our CHO-based CFPS platform are attained within 2 days and it is well-suited for automation. Further development would provide a tool for rapid, high-throughput prediction of expression ranking of mAb producers to accelerate design-build-test cycles required for antibody expression and engineering. Looking forward, the CHO-based CFPS platform could facilitate the synthesis of toxic proteins as well.
Foodborne bacterial infections and diseases have been considered to be a major threat for public health in the worldwide. Increased incidence of human diseases caused by foodborne pathogens have been correlated with growing world population and mobility. Loop-mediated isothermal amplification (LAMP) has been regarded as an innovative gene amplification technology and emerged as an alternative to PCR-based methodologies in both clinical laboratory and food safety testing. Nowadays, LAMP has been applied to detection and identification on pathogens from microbial diseases, as it showed significant advantage in high sensitivity, specificity and rapidity. The high sensitivity of LAMP enables detection of the pathogens in sample materials even without time consuming sample preparation. An overview of LAMP mainly containing the development history, reaction principle and its application to four kind of foodborne pathogens detection are presented in this paper. As concluded, with the advantages of rapidity, simplicity, sensitivity, specificity and robustness, LAMP is capable of applications for clinical diagnosis as well as surveillance of infection diseases. Moreover, the main purpose of this paper is to provide theoretical basis for the clinical application of LAMP technology.
Streptomyces venezuelae is a promising chassis in synthetic biology for fine chemical and secondary metabolite pathway engineering. The potential of S. venezuelae could be further realized by expanding its capability with the introduction of its own in vitro transcription-translation (TX-TL) system. TX-TL is a fast and expanding technology for bottom-up design of complex gene expression tools, biosensors and protein manufacturing. Herein, we introduce a S. venezuelae TX-TL platform by reporting a streamlined protocol for cell-extract preparation, demonstrating high-yield synthesis of a codon-optimized sfGFP reporter and the prototyping of a synthetic tetracycline-inducible promoter in S. venezuelae TX-TL based on the TetO-TetR repressor system. The aim of this system is to provide a host for the homologous production of exotic enzymes from Actinobacteria secondary metabolism in vitro. As an example, we demonstrate the soluble synthesis of a selection of enzymes (12-70 kDa) from the Streptomyces rimosus oxytetracycline pathway.
Messenger RNA (mRNA) plays a critical role in cellular growth and development. However, there have been limited methods available to visualize endogenous mRNA in living cells with ease. We have designed RNA-based fluorescence “turn-on” probes that target mRNA by fusing an unstable form of Spinach with target-complementary sequences. These probes have been demonstrated to be selective, stable and capable of targeting various mRNAs for live E. coli imaging.