Cell-free Expression System Research Articles

The role of lipid bilayer composition in cell-free expressed ABC transporter folding.

Cell-free protein expression systems provide a powerful tool to mechanistically probe how membrane composition, and the resulting biophysical properties, affect membrane protein (MP) complex folding and function. These systems can be designed without translocons, insertases, and chaperones commonly used to assist in membrane protein folding to isolate the impact of membrane properties on the folding process. In this study we investigate the ABC transporter, SapABCDF, in the gram-negative non-typeable Haemophilus influenzae (NTHI). This transporter sequesters heme and antimicrobial peptides (AMPs) from the periplasm into the cytoplasm via ATPase activity and understanding factors that contribute to MP complex folding and function is critical for developing antimicrobial treatments for NTHI and reduce the severity of infection. Using a cell-free protein expression system, we expressed each protein in the SapABCDF transporter in the presence of small unilamellar lipid vesicles (SUVs). We developed a sucrose flotation assay to evaluate protein colocalization to fluorescently labeled SUVs after cell-free expression and found that all components of the SapBCDF system express and colocalize to vesicles, suggesting their membrane integration and assembly. We then conducted a co-translational MP folding assay with a C-terminal mEGFP tag to determine membrane compositions that promote MP folding and expression. Analysis of cell-free expression products via western blot suggests that one transmembrane domain, SapC, expresses preferentially into SUVs containing phosphatidylethanolamine (PE). Ongoing studies aim to characterize transporter functionality and biophysically characterize the impact of bilayer biophysical properties on MP folding. Together, our results should improve our understanding of the biophysical requirements for properly folded and functioning ABC transporters.

Tuning the composition of supported lipid bilayers in a QCMD to uncover rules of membrane protein incorporation.

Cells are not very cooperative when it comes to helping us understand them better. They are small, needy, complex, and they have their own lives to run. To circumvent these flaws of cells our lab has developed the all E. coli cell-free protein expression system called TXTL. TXTL can express genes and execute genetic programs in-vitro using the transcriptional and translational machinery of E. coli cells without the E. coli cells themselves. So far with TXTL our lab has probed both fundamental and applied ideas in gene regulation, metabolism, self-assembly, and encapsulation.

Cell-Free Dot Blot: an Ultra-Low-Cost and Practical Immunoassay Platform for Detection of Anti-SARS-CoV-2 Antibodies in Human and Animal Sera.

Since its emergence in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has caused severe disruption to key aspects of human life globally and highlighted the need for timely, adaptive, and accessible pandemic response strategies. Here, we introduce the cell-free dot blot (CFDB) method, a practical and ultra-low-cost immune diagnostic platform capable of rapid response and mass immunity screening for the current and future pandemics. Similar in mechanism to the widely used enzyme-linked immunosorbent assays (ELISAs), our method is novel and advantageous in that (i) it uses linear DNA to produce the target viral antigen fused to a SpyTag peptide in a cell-free expression system without the need for traditional cloning and antigen purification, (ii) it uses SpyCatcher2-Apex2, an Escherichia coli-produced peroxidase conjugate as a universal secondary detection reagent, obviating the need for commercial or sophisticated enzyme conjugates, and (iii) sera are spotted directly on a nitrocellulose membrane, enabling a simple "dipping" mechanism for downstream incubation and washing steps, as opposed to individual processing of wells in a multiwell plate. To demonstrate the utility of our method, we performed CFDB to detect anti-severe acute respiratory syndrome coronavirus 2 nucleocapsid protein antibodies in precharacterized human sera (23 negative and 36 positive for COVID-19) and hamster sera (16 negative and 36 positive for COVID-19), including independent testing at a collaborating laboratory, and we show assay performance comparable to that of conventional ELISAs. At a similar capacity to 96-well plate ELISA kits, one CFDB assay costs only ~$3 USD. We believe that CFDB can become a valuable pandemic response tool for adaptive and accessible sero-surveillance in human and animal populations. IMPORTANCE The recent COVID-19 pandemic has highlighted the need for diagnostic platforms that are rapidly adaptable, affordable, and accessible globally, especially for low-resource settings. To address this need, we describe the development and functional validation of a novel immunoassay technique termed the cell-free dot blot (CFDB) method. Based on the principles of cell-free synthetic biology and alternative dot blotting procedures, our CFDB immunoassay is designed to provide for timely, practical, and low-cost responses to existing and emerging public health threats, such as the COVID-19 pandemic, at a similar throughput and comparable performance as conventional ELISAs. Notably, the molecular detection reagents used in CFDB can be produced rapidly in-house, using established protocols and basic laboratory infrastructure, minimizing reliance on strained commercial reagents. In addition, the materials and imaging instruments required for CFDB are the same as those used for common Western blotting experiments, further expanding the reach of CFDB in decentralized facilities.

Characterization of Integrase and Excisionase Activity in a Cell-Free Protein Expression System Using a Modeling and Analysis Pipeline.

We present a full-stack modeling, analysis, and parameter identification pipeline to guide the modeling and design of biological systems starting from specifications to circuit implementations and parametrizations. We demonstrate this pipeline by characterizing the integrase and excisionase activity in a cell-free protein expression system. We build on existing Python tools─BioCRNpyler, AutoReduce, and Bioscrape─to create this pipeline. For enzyme-mediated DNA recombination in a cell-free system, we create detailed chemical reaction network models from simple high-level descriptions of the biological circuits and their context using BioCRNpyler. We use Bioscrape to show that the output of the detailed model is sensitive to many parameters. However, parameter identification is infeasible for this high-dimensional model; hence, we use AutoReduce to automatically obtain reduced models that have fewer parameters. This results in a hierarchy of reduced models under different assumptions to finally arrive at a minimal ODE model for each circuit. Then, we run sensitivity analysis-guided Bayesian inference using Bioscrape for each circuit to identify the model parameters. This process allows us to quantify integrase and excisionase activity in cell extracts enabling complex-circuit designs that depend on accurate control over protein expression levels through DNA recombination. The automated pipeline presented in this paper opens up a new approach to complex circuit design, modeling, reduction, and parametrization.

Cell-Free Synthesis Goes Electric: Dual Optical and Electronic Biosensor via Direct Channel Integration into a Supported Membrane Electrode.

Assembling transmembrane proteins on organic electronic materials is one promising approach to couple biological functions to electrical readouts. A biosensing device produced in such a way would enable both the monitoring and regulation of physiological processes and the development of new analytical tools to identify drug targets and new protein functionalities. While transmembrane proteins can be interfaced with bioelectronics through supported lipid bilayers (SLBs), incorporating functional and oriented transmembrane proteins into these structures remains challenging. Here, we demonstrate that cell-free expression systems allow for the one-step integration of an ion channel into SLBs assembled on an organic conducting polymer, poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS). Using the large conductance mechanosensitive channel (MscL) as a model ion channel, we demonstrate that MscL adopts the correct orientation, remains mobile in the SLB, and is active on the polyelectrolyte surface using optical and electrical readouts. This work serves as an important illustration of a rapidly assembled bioelectronic platform with a diverse array of downstream applications, including electrochemical sensing, physiological regulation, and screening of transmembrane protein modulators.

Electrochemical studies of the mitochondrial ROMK2 potassium channel activity reconstituted into the free-standing and tethered bilayer lipid membranes

The renal-outer-medullary‑potassium (ROMK2) channel modulates potassium transport in the kidney. It has been postulated that the ROMK2 is the pore-forming subunit of the mitochondrial ATP-sensitive potassium channel as a mediator of cardioprotection. In this study, cell-free synthesis of the ROMK2 was performed in presence of membrane scaffold protein (MSP1D1) nanodiscs. Activity measurements were achieved after channel reconstitution into the planar lipid bilayer and tethered bilayer lipid membranes. Both methods allowed for monitoring of channel function, verified with channel blocking and activation/re-activation experiments. The primary function of the mitochondrial potassium channels is to regulate the potential of the mitochondrial membrane, which allows them to play an important role in cytoprotection. This work focuses on obtaining the ROMK2 using a cell-free expression system, followed by the incorporation of the channel protein into the lipid bilayer and studying the influence of voltage changes and molecular modulators on channel activity. Channel activity was measured after its reconstitution into two models of lipid bilayers - BLM (Bilayer Lipid Membrane) and tBLM (Tethered Bilayer Lipid Membrane) deposited on a solid gold electrode. These two model membranes and electrochemical measurements made it possible to measure the flux of K+ ions in the presence of channel modulators.

Compartmentalized Cell-Free Expression Systems for Building Synthetic Cells.

One of the grand challenges in bottom-up synthetic biology is the design and construction of synthetic cellular systems. One strategy toward this goal is the systematic reconstitution of biological processes using purified or non-living molecular components to recreate specific cellular functions such as metabolism, intercellular communication, signal transduction, and growth and division. Cell-free expression systems (CFES) are in vitro reconstitutions of the transcription and translation machinery found in cells and are a key technology for bottom-up synthetic biology. The open and simplified reaction environment of CFES has helped researchers discover fundamental concepts in the molecular biology of the cell. In recent decades, there has been a drive to encapsulate CFES reactions into cell-like compartments with the aim of building synthetic cells and multicellular systems. In this chapter, we discuss recent progress in compartmentalizing CFES to build simple and minimal models of biological processes that can help provide a better understanding of the process of self-assembly in molecularly complex systems.

Rapid and Finely-Tuned Expression for Deployable Sensing Applications.

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.

Programmable regulation of translation by harnessing the CRISPR-Cas13 system.

The ability to control protein expression at both the transcriptional and post-transcriptional levels is instrumental for the cell to integrate multiple molecular signals and then reach high operational sophistication. Although challenging, fully artificial regulations at different levels are required for boosting systems and synthetic biology. Here, we report the development of a novel framework to regulate translation by repurposing the CRISPR-Cas13 immune system, which uses an RNA-guided ribonuclease. By exploiting a cell-free expression system for prototyping gene regulatory structures, our results demonstrate that CRISPR-dCas13a ribonucleoproteins (d means catalytically dead) can be programmed to repress or activate translation initiation. The performance assessment of the engineered systems also revealed guide RNA design principles. Moreover, we show that the system can work in vivo. This development complements the ability to regulate transcription with other CRISPR-Cas systems and offers potential applications.

Activation/Inhibition of Gene Expression Caused by Alcohols: Relationship with the Viscoelastic Property of a DNA Molecule

Alcohols are used in the life sciences because they can condense and precipitate DNA. Alcohol consumption has been linked to many diseases and can alter genetic activity. In the present report, we carried out experiments to make clear how alcohols affect the efficiency of transcription-translation (TX-TL) and translation (TL) by adapting cell-free gene expression systems with plasmid DNA and RNA templates, respectively. In addition, we quantitatively analyzed intrachain fluctuations of single giant DNA molecules based on the fluctuation-dissipation theorem to gain insight into how alcohols affect the dynamical property of a DNA molecule. Ethanol (2-3%) increased gene expression levels four to five times higher than the control in the TX-TL reaction. A similar level of enhancement was observed with 2-propanol, in contrast to the inhibitory effect of 1-propanol. Similar alcohol effects were observed for the TL reaction. Intrachain fluctuation analysis through single DNA observation showed that 1-propanol markedly increased both the spring and damping constants of single DNA in contrast to the weak effects observed with ethanol, whereas 2-propanol exhibits an intermediate effect. This study indicates that the activation/inhibition effects of alcohol isomers on gene expression correlate with the changes in the viscoelastic mechanical properties of DNA molecules.

Detection of Bile Acids in Complex Matrices Using a Transcription Factor-Based Biosensor.

Bile acids play an important role in digestion and human health, are found throughout the gastrointestinal tract, and are excreted in feces. Therefore, bile acids are promising biomarkers for monitoring health and detecting fecal contamination in water sources. Here, we engineered a bile acid sensor by expressing the transcription factor BreR, a TetR-like repressor from Vibrio cholorae, in Escherichia coli. The sensor was further optimized by screening a promoter library. To further characterize the BreR sensor and increase its utility, we moved expression to a cell-free expression (CFE) system, resulting in an approximately 3 orders of magnitude increase in deoxycholic acid sensitivity. We next optimized this sensor to detect bile acids in fecal water, wastewater, and serum and transferred the CFE sensor to a paper-based assay to enhance fieldability.

A novel metagenome-derived viral RNA polymerase and its application in a cell-free expression system for metagenome screening

The mining of genomes from non-cultivated microorganisms using metagenomics is a powerful tool to discover novel proteins and other valuable biomolecules. However, function-based metagenome searches are often limited by the time-consuming expression of the active proteins in various heterologous host systems. We here report the initial characterization of novel single-subunit bacteriophage RNA polymerase, EM1 RNAP, identified from a metagenome data set obtained from an elephant dung microbiome. EM1 RNAP and its promoter sequence are distantly related to T7 RNA polymerase. Using EM1 RNAP and a translation-competent Escherichia coli extract, we have developed an efficient medium-throughput pipeline and protocol allowing the expression of metagenome-derived genes and the production of proteins in cell-free system is sufficient for the initial testing of the predicted activities. Here, we have successfully identified and verified 12 enzymes acting on bis(2-hydroxyethyl) terephthalate (BHET) in a completely clone-free approach and proposed an in vitro high-throughput metagenomic screening method.

<em>In Vitro</em> Cleavage Assays using Purified Recombinant <em>Drosophila</em> Caspases for Substrate Screening

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis has been very well defined and many apoptotic proteolytic substrates of caspases have been identified and characterized, the role of caspases for non-apoptotic processes is not well understood. In particular, few non-apoptotic substrates of caspases have been identified thus far. Here, in order to facilitate the identification and characterization of potential caspase substrates, a protocol that allows the testing of candidate substrates in caspase cleavage assays in vitro is described. This protocol includes the production and purification of recombinant caspase proteins, the production of the candidate substrates either recombinantly or in a cell-free expression system, and the actual in vitro cleavage reaction followed by SDS-PAGE and immunoblotting. This protocol is tailored for the Drosophila caspases Dronc and Drice but can easily be adapted for caspases from other organisms, including mammals.

In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening.

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis has been very well defined and many apoptotic proteolytic substrates of caspases have been identified and characterized, the role of caspases for non-apoptotic processes is not well understood. In particular, few non-apoptotic substrates of caspases have been identified thus far. Here, in order to facilitate the identification and characterization of potential caspase substrates, a protocol that allows the testing of candidate substrates in caspase cleavage assays in vitro is described. This protocol includes the production and purification of recombinant caspase proteins, the production of the candidate substrates either recombinantly or in a cell-free expression system, and the actual in vitro cleavage reaction followed by SDS-PAGE and immunoblotting. This protocol is tailored for the Drosophila caspases Dronc and Drice but can easily be adapted for caspases from other organisms, including mammals.

Phospholipid synthesis inside phospholipid membrane vesicles

Construction of living artificial cells from genes and molecules can expand our understanding of life system and establish a new aspect of bioengineering. However, growth and division of cell membrane that are basis of cell proliferation are still difficult to reconstruct because a high-yielding phospholipid synthesis system has not been established. Here, we developed a cell-free phospholipid synthesis system that combines fatty acid synthesis and cell-free gene expression system synthesizing acyltransferases. The synthesized fatty acids were sequentially converted into phosphatidic acids by the cell-free synthesized acyltransferases. Because the system can avoid the accumulation of intermediates inhibiting lipid synthesis, sub-millimolar phospholipids could be synthesized within a single reaction mixture. We also performed phospholipid synthesis inside phospholipid membrane vesicles, which encapsulated all the components, and showed the phospholipids localized onto the mother membrane. Our approach would be a platform for the construction of self-reproducing artificial cells since the membrane can grow sustainably.

A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems.

Cell-free gene expression (CFE) systems are an attractive tool for engineering within synthetic biology and for industrial production of high-value recombinant proteins. CFE reactions require a cell extract, energy system, amino acids, and DNA, to catalyse mRNA transcription and protein synthesis. To provide an amino acid source, CFE systems typically use a commercial standard, which is often proprietary. Herein we show that a range of common microbiology rich media (i.e., tryptone, peptone, yeast extract and casamino acids) unexpectedly provide an effective and low-cost amino acid source. We show that this approach is generalisable, by comparing batch variability and protein production in the following range of CFE systems: Escherichia coli (Rosetta™ 2 (DE3), BL21(DE3)), Streptomyces venezuelae and Pichia pastoris. In all CFE systems, we show equivalent or increased protein synthesis capacity upon replacement of the commercial amino acid source. In conclusion, we suggest rich microbiology media provides a new amino acid source for CFE systems with potential broad use in synthetic biology and industrial biotechnology applications.

Steady-State Operation of a Cell-Free Genetic Band-Detection Circuit.

Pattern formation processes play a decisive role during embryogenesis and involve the precise spatial and temporal orchestration of intricate gene regulatory processes. Synthetic gene circuits modeled after their biological counterparts can be used to investigate such processes under well-controlled conditions and may, in the future, be utilized for autonomous position determination in synthetic biological materials. Here, we investigated a three-node feed-forward gene regulatory circuit in vitro that generates three distinct fluorescent outputs in response to varying concentrations of a single externally supplied morphogen. The circuit acts as a band detector for the morphogen concentration and, in a spatial context, could serve as a stripe-forming gene circuit. We simulated the behavior of the genetic circuit in the presence of a morphogen gradient using a system of ordinary differential equations and determined optimal parameters for stripe-pattern formation using an evolutionary algorithm. To analyze the subcircuits of the system, we conducted cell-free characterization experiments and finally tested the whole genetic circuit in nanoliter-scale microfluidic flow reactors that provided a continuous supply of cell extract and metabolites and allowed removal of degradation products. To make use of the widely employed promoters PLlacO-1 and PLtetO-1 in our design, we removed LacI from our bacterial cell extract by genome engineering Escherichia coli using a pORTMAGE workflow. Our results show that the band-detector works as designed when operated out of equilibrium within the flow reactors. On the other hand, subcircuits of the system and the whole circuit fail to generate the desired gene expression response when operated in a closed reactor. Our work thus underlines the importance of out-of-equilibrium operation of complex gene circuits, which cannot settle to a steady-state expression pattern within the finite lifetime of a cell-free expression system.

Effects of DNA template preparation on variability in cell-free protein production.

DNA templates for protein production remain an unexplored source of variability in the performance of cell-free expression (CFE) systems. To characterize this variability, we investigated the effects of two common DNA extraction methodologies, a postprocessing step and manual versus automated preparation on protein production using CFE. We assess the concentration of the DNA template, the quality of the DNA template in terms of physical damage and the quality of the DNA solution in terms of purity resulting from eight DNA preparation workflows. We measure the variance in protein titer and rate of protein production in CFE reactions associated with the biological replicate of the DNA template, the technical replicate DNA solution prepared with the same workflow and the measurement replicate of nominally identical CFE reactions. We offer practical guidance for preparing and characterizing DNA templates to achieve acceptable variability in CFE performance.

Rapid engineering of surface acoustic wave devices using cell-free expressed bitter receptors towards chemical sensing

The engineering of devices with various functional molecules in a high-efficient and convenient manner is crucial for biosensors towards biomedical applications. In this study, a multi-channel surface acoustic wave (SAW) device was rapid engineered with bitter receptors prepared from a cell-free protein expression system for the development of biomimetic biosensors for the detection of bitter substances. The novelty of this study mainly attributed to the utilization of DNA-directed immobilization (DDI) method to achieve high-efficient and controllable immobilization of bitter receptors onto sensitive area of SAW device. SAW device is employed as a transducer to monitor the specific interactions between bitter receptors and their ligands, which is a mass-sensitive device that can monitor tiny mass changes by recording the changes in the phase shift of SAW. The obtained results indicated that this biomimetic biosensor was able to respond to different bitter substances by generating various responsive signals, which show good stability. In addition, the distinct responsive patterns could be used to discriminating different bitter substances. It was successfully used to detect and discriminate 10 μM MgSO4, 0.1 μM denatonium, 0.1 μM D-(−)-Salicin, and 0.1 μM quinine. This biomimetic biosensor has great potential to be used as a novel tool for chemical sensing as well as for the research of mechanisms of bitter receptor interacting with their ligands.

A Functional Carbohydrate Degrading Enzyme Potentially Acquired by Horizontal Gene Transfer in the Genome of the Soil Invertebrate Folsomia candida.

Horizontal gene transfer (HGT) is defined as the acquisition by an organism of hereditary material from a phylogenetically unrelated organism. This process is mostly observed among bacteria and archaea, and considered less likely between microbes and multicellular eukaryotes. However, recent studies provide compelling evidence of the evolutionary importance of HGT in eukaryotes, driving functional innovation. Here, we study an HGT event in Folsomia candida (Collembola, Hexapoda) of a carbohydrate-active enzyme homologous to glycosyl hydrase group 43 (GH43). The gene encodes an N-terminal signal peptide, targeting the product for excretion, which suggests that it contributes to the diversity of digestive capacities of the detritivore host. The predicted α-L-arabinofuranosidase shows high similarity to genes in two other Collembola, an insect and a tardigrade. The gene was cloned and expressed in Escherichia coli using a cell-free protein expression system. The expressed protein showed activity against p-nitrophenyl-α-L-arabinofuranoside. Our work provides evidence for functional activity of an HGT gene in a soil-living detritivore, most likely from a bacterial donor, with genuine eukaryotic properties, such as a signal peptide. Co-evolution of metazoan GH43 genes with the Panarthropoda phylogeny suggests the HGT event took place early in the evolution of this ecdysozoan lineage.