Cell-free Transcription-translation System Research Articles

Design and Characterization of a Gene-Encoding DNA Nanoparticle in a Cell-Free Transcription–Translation System

Design and Characterization of a Gene-Encoding DNA Nanoparticle in a Cell-Free Transcription–Translation System

Toward Minimal Transcription-Translation Machinery.

With the advantages of simple genetic composition, low metabolic background, low energy waste, and high genetic stability, genome-reduced strains, as promising functional chassis, have become an intensive direction for constructing potent biosynthesis factories. Herein, an innovative Genome-Reduced strain-based Active Cell-free Easy-to-make-protein (GRACE) system is built as minimal transcription-translation machinery. In this study, two Escherichia coli genome-reduced strains, ΔW3110 and ΔMG1655, with genome reduction of 11.53% and 37.85%, are fused with the cell-free transcription-translation (CFTT) system. The GRACE systems perform better than the corresponding CFTT systems derived from their parental strains in representative valuable applications, such as the expression and solubilization of membrane proteins or protein polymers, biosensing of inorganic or organic molecules based on different principles, and unnatural amino acid embedding. Obviously, the GRACE system has provided a brand-new enabling platform for cell-free transcription-translation basic and applied studies and also would inspire the potential of genome-reduced strains for versatile applications.

Markedly Different Effects of Monovalent Cations on the Efficiency of Gene Expression.

The effect of monovalent cations on a cell-free transcription-translation (TX-TL) system is examined using a luciferase assay. It is found that the potency for all ions analyzed here is in the order Rb+ >K+ >Cs+ > Na+ ≈Li+ >(CH3 )4 N+ , where Rb+ is most efficient at promoting TX-TL and the ions of Li+ , Na+ , and (CH3 )4 N+ exhibit an inhibitory effect. Similar promotion/inhibition effects are observed for cell-free TL alone with an mRNA template.

A TXTL-Based Assay to Rapidly Identify PAMs for CRISPR-Cas Systems with Multi-Protein Effector Complexes.

Type I CRISPR-Cas systems represent the most common and diverse type of these prokaryotic defense systems and are being harnessed for a growing set of applications. As these systems rely on multi-protein effector complexes, their characterization remains challenging. Here, we report a rapid and straightforward method to characterize these systems in a cell-free transcription-translation (TXTL) system. A ribonucleoprotein complex is produced and binds to its target next to a recognized PAM, thereby preventing the targeted sequence from being cleaved by a restriction enzyme. Selection for uncleaved targeted plasmids leads to an enrichment of recognized sequences within a PAM library. This assay will aid the exploration of CRISPR-Cas diversity and evolution and help contribute new systems for CRISPR technologies and applications.

Rapid screening of glycosyltransferases in plants using a linear DNA expression template based cell-free transcription-translation system

Plants have an extensively large number of enzymes including glycosyltransferases that are important in the biosynthesis of natural products. However, it is time-consuming and challenging to study these enzymes and only a small percentage of them have been well-characterized. Here, we report a rapid method to screen plant glycosyltransferases using a linear DNA expression template (LET) based cell-free transcription-translation system (TX-TL). As a proof of concept, we amplified and tested glycosyltransferases from Arabidopsis thaliana and showed that the catalytic activity results of these glycosyltransferases from LET-based-TX-TL were consistent with previous studies. We then chose a local medicinal plant Anoectochilus roxburghii, acquired its transcriptome sequences, and applied this method to study its glycosyltransferases. We rapidly expressed all the putative UDP-glucose glycosyltransferases using LET-based-TX-TL and discovered 6 unreported active glycosyltransferases which can catalyze the glycosylation of quercetin into isoquercitrin. Thus, LET-based-TX-TL was shown to be a powerful tool for researchers to rapidly screen plant glycosyltransferases for the first time.

Expression of Fatty Acyl-CoA Ligase Drives One-Pot De Novo Synthesis of Membrane-Bound Vesicles in a Cell-Free Transcription-Translation System.

Despite the central importance of lipid membranes in cellular organization, it is challenging to reconstitute their formation de novo from minimal chemical and biological elements. Here, we describe a chemoenzymatic route to membrane-forming noncanonical phospholipids in which cysteine-modified lysolipids undergo spontaneous coupling with fatty acyl-CoA thioesters generated enzymatically by a fatty acyl-CoA ligase. Due to the high efficiency of the reaction, we were able to optimize phospholipid formation in a cell-free transcription-translation (TX-TL) system. Combining DNA encoding the fatty acyl-CoA ligase with suitable lipid precursors enabled one-pot de novo synthesis of membrane-bound vesicles. Noncanonical sphingolipid synthesis was also possible by using a cysteine-modified lysosphingomyelin as a precursor. When the sphingomyelin-interacting protein lysenin was coexpressed alongside the acyl-CoA ligase, the in situ assembled membranes were spontaneously decorated with protein. Our strategy of coupling gene expression with membrane lipid synthesis in a one-pot fashion could facilitate the generation of proteoliposomes and brings us closer to the bottom-up generation of synthetic cells using recombinant synthetic biology platforms.

Cell-Free Characterization of Coherent Feed-Forward Loop-Based Synthetic Genetic Circuits.

Regulatory pathways inside living cells employ feed-forward architectures to fulfill essential signal processing functions that aid in the interpretation of various types of inputs through noise-filtering, fold-change detection and adaptation. Although it has been demonstrated computationally that a coherent feed-forward loop (CFFL) can function as noise filter, a property essential to decoding complex temporal signals, this motif has not been extensively characterized experimentally or integrated into larger networks. Here we use post-transcriptional regulation to implement and characterize a synthetic CFFL in an Escherichia coli cell-free transcription-translation system and build larger composite feed-forward architectures. We employ microfluidic flow reactors to probe the response of the CFFL circuit using both persistent and short, noise-like inputs and analyze the influence of different circuit components on the steady-state and dynamics of the output. We demonstrate that our synthetic CFFL implementation can reliably repress background activity compared to a reference circuit, but displays low potential as a temporal filter, and validate these findings using a computational model. Our results offer practical insight into the putative noise-filtering behavior of CFFLs and show that this motif can be used to mitigate leakage and increase the fold-change of the output of synthetic genetic circuits.

Sequence-independent RNA sensing and DNA targeting by a split domain CRISPR-Cas12a gRNA switch.

CRISPR technologies increasingly require spatiotemporal and dosage control of nuclease activity. One promising strategy involves linking nuclease activity to a cell's transcriptional state by engineering guide RNAs (gRNAs) to function only after complexing with a ‘trigger’ RNA. However, standard gRNA switch designs do not allow independent selection of trigger and guide sequences, limiting gRNA switch application. Here, we demonstrate the modular design of Cas12a gRNA switches that decouples selection of these sequences. The 5′ end of the Cas12a gRNA is fused to two distinct and non-overlapping domains: one base pairs with the gRNA repeat, blocking formation of a hairpin required for Cas12a recognition; the other hybridizes to the RNA trigger, stimulating refolding of the gRNA repeat and subsequent gRNA-dependent Cas12a activity. Using a cell-free transcription-translation system and Escherichia coli, we show that designed gRNA switches can respond to different triggers and target different DNA sequences. Modulating the length and composition of the sensory domain altered gRNA switch performance. Finally, gRNA switches could be designed to sense endogenous RNAs expressed only under specific growth conditions, rendering Cas12a targeting activity dependent on cellular metabolism and stress. Our design framework thus further enables tethering of CRISPR activities to cellular states.

Specificities and functional coordination between the two Cas6 maturation endonucleases in Anabaena sp. PCC 7120 assign orphan CRISPR arrays to three groups

ABSTRACT Many bacteria and archaea possess an RNA-guided adaptive and inheritable immune system that consists of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. In most CRISPR-Cas systems, the maturation of CRISPR-derived small RNAs (crRNAs) is essential for functionality. Cas6 endonucleases function as the most frequent CRISPR RNA maturation enzymes. In the cyanobacterium Anabaena sp. PCC 7120, ten CRISPR loci are present, but only two cas gene cassettes plus a Tn7-associated eleventh array. In this study, we deleted the two cas6 genes alr1482 (Type III-D) or alr1566 (Type I-D) and tested the specificities of the two corresponding enzymes in the resulting mutant strains, as recombinant proteins and in a cell-free transcription-translation system. The results assign the direct repeats (DRs) to three different groups. While Alr1566 is specific for one group, Alr1482 has a higher preference for the DRs of the second group but can also cleave those of the first group. We found that this cross-recognition limits crRNA accumulation for the Type I-D system in vivo. We also show that the DR of the cas gene-free CRISPR array of cyanophage N-1 is processed by these enzymes, suggesting that it is fully competent in association with host-encoded Cas proteins. The data support the functionality of CRISPR arrays that frequently appear fragmented to multiple genomic loci in multicellular cyanobacteria and disfavour other possibilities, such as the nonfunctionality of these orphan repeat-spacer arrays. Our results show the functional coordination of Cas6 endonucleases with both neighbouring and remote repeat-spacer arrays in the CRISPR-Cas system of cyanobacteria.

Detection of Human p53 In-Vitro Expressed in a Transcription-Translation Cell-Free System by a Novel Conjugate Based on Cadmium Sulphide Nanoparticles.

Here, cadmium sulphide quantum dots (CdS QDs) have been synthetized and functionalized with Bovine Serum Albumin (BSA) in a colloidal aqueous solution with a stability of over 3 months. Specific synthesis conditions, in homogeneous phase and at low temperature, have allowed limitation of S2− concentration, hence, as a consequence, there is restricted growth of the nanoparticles (NPs). This fact allows binding with BSA in the most favorable manner for the biomolecule. The presence of Cd2+ ions on the surface of the CdS nanoparticle is counteracted by the negatively charged domains of the BSA, resulting in the formation of small NPs, with little tendency for aggregation. Temperature and pH have great influence on the fluorescence characteristics of the synthetized nanoparticles. Working at low temperatures (4 °C) and pH 10–11 have proven the best result as shown by hydrolysis kinetic control of the thioacetamide precursor of S2− ion. Biological activity of the coupled BSA is maintained allowing subsequent bioconjugation with other biomolecules such as antibodies. The chemical conjugation with anti-Glutathione S-transferase (α-GST) antibody, a common tag employed in human recombinant fusion proteins, produces a strong quenching of fluorescence that proves the possibilities of its use in biological labelling. Finally, p53, onco-human recombinant protein (GST tagged in COOH terminus), has been in situ IVTT (in vitro transcription-translation) expressed and efficiently captured by the α-GST-CdS QD conjugate as a proof of the biocompatibility on IVTT systems and the functionality of conjugated antibody.

In vitro implementation of robust gene regulation in a synthetic biomolecular integral controller

Feedback mechanisms play a critical role in the maintenance of cell homeostasis in the presence of disturbances and uncertainties. Motivated by the need to tune the dynamics and improve the robustness of gene circuits, biological engineers have proposed various designs that mimic natural molecular feedback control mechanisms. However, practical and predictable implementations have proved challenging because of the complexity of synthesis and analysis of complex biomolecular networks. Here, we analyze and experimentally validate a synthetic biomolecular controller executed in vitro. The controller ensures that gene expression rate tracks an externally imposed reference level, and achieves this goal even in the presence of certain kinds of disturbances. Our design relies upon an analog of the well-known principle of integral feedback in control theory. We implement the controller in an Escherichia coli cell-free transcription-translation system, which allows rapid prototyping and implementation. Modeling and theory guide experimental implementation with well-defined operational predictability.

Cell-Free Prototyping of AND-Logic Gates Based on Heterogeneous RNA Activators.

RNA-based devices controlling gene expression bear great promise for synthetic biology, as they offer many advantages such as short response times and light metabolic burden compared to protein-circuits. However, little work has been done regarding their integration to multilevel regulated circuits. In this work, we combined a variety of small transcriptional activator RNAs (STARs) and toehold switches to build highly effective AND-gates. To characterize the components and their dynamic range, we used an Escherichia coli (E. coli) cell-free transcription-translation (TX-TL) system dispensed via nanoliter droplets. We analyzed a prototype gate in vitro as well as in silico, employing parametrized ordinary differential equations (ODEs), for which parameters were inferred via parallel tempering, a Markov chain Monte Carlo (MCMC) method. On the basis of this analysis, we created nine additional AND-gates and tested them in vitro. The functionality of the gates was found to be highly dependent on the concentration of the activating RNA for either the STAR or the toehold switch. All gates were successfully implemented in vivo, offering a dynamic range comparable to the level of protein circuits. This study shows the potential of a rapid prototyping approach for RNA circuit design, using cell-free systems in combination with a model prediction.

An Adaptive Synthetic Cell Based on Mechanosensing, Biosensing, and Inducible Gene Circuits.

The bottom-up assembly of synthetic cell systems capable of recapitulating biological functions has become a means to understand living matter by construction. The integration of biomolecular components into active, cell-sized, genetically programmed compartments remains, however, a major bottleneck for building synthetic cells. A primary feature of real cells is their ability to actively interact with their surroundings, particularly in stressed conditions. Here, we construct a synthetic cell equipped with an inducible genetic circuit that responds to changes in osmotic pressure through the mechanosensitive channel MscL. Liposomes loaded with an E.coli cell-free transcription-translation (TXTL) system are induced with IPTG when exposed to hypo-osmotic solution, resulting in the expression of a bacterial cytoskeletal protein MreB. MreB associates with the membrane to generate a cortex-like structure. Our work provides the first example of molecular integration that couples mechanosensitivity, gene expression, and self-assembly at the inner membrane of synthetic cells.

An enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system

The characterization of CRISPR-Cas immune systems in bacteria was quickly followed by the discovery of anti-CRISPR proteins (Acrs) in bacteriophages. These proteins block different steps of CRISPR-based immunity and, as some inhibit Cas nucleases, can offer tight control over CRISPR technologies. While Acrs have been identified against a few CRISPR-Cas systems, likely many more await discovery and application. Here, we report a rapid and scalable method for characterizing putative Acrs against Cas nucleases using an E. coli-derived cell-free transcription-translation system. Using known Acrs against type II Cas9 nucleases as models, we demonstrate how the method can be used to measure the inhibitory activity of individual Acrs in under two days. We also show how the method can overcome non-specific inhibition of gene expression observed for some Acrs. In total, the method should accelerate the interrogation and application of Acrs as CRISPR-Cas inhibitors.

Construction of Incoherent Feedforward Loop Circuits in a Cell-Free System and in Cells.

Cells utilize transcriptional regulation networks to respond to environmental signals. Network motifs, such as feedforward loops, play essential roles in these regulatory networks. In this work, we construct two different functional and modular incoherent type 1 feedforward loop circuits in a cell-free transcription-translation system and in cells. With the help of mathematical modeling and the cell-free system, we can streamline the design-build-test cycles of the circuits, in which we characterize and optimize these circuits in vitro to confirm that they function as expected before implementing them in vivo. We show that the performance of these circuits from in vitro studies closely recapitulates those from in vivo experiments. We demonstrate that these feedforward loops show dynamic response and pulse-like behavior both in vitro and in vivo. These novel feedforward loop network motifs can be incorporated in more complicated biological circuits as detectors or responders.

Distinct timescales of RNA regulators enable the construction of a genetic pulse generator.

To build complex genetic networks with predictable behaviors, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher-order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely used to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA-based transcriptional regulation, using small transcription activating RNAs (STARs) and clustered regularly interspaced short palindromic repeats interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell-free transcription-translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ordinary differential equation-based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modeling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity.

On-Demand Production of Femtoliter Drops in Microchannels and Their Use as Biological Reaction Compartments.

We present a method allowing to produce monodisperse droplets with volumes in the femtoliter range in a microchannel on demand. The method utilizes pulsed electric fields deforming the interface between an aqueous and an oil phase and pinching off droplets. Water and xanthan gum solutions are considered as disperse-phase liquids, and it is shown that the method can be applied even to solutions with a zero-shear rate viscosity more than 104-times higher than that of water. The droplet formation regimes are explored by systematically varying the pulse amplitude and duration as well as the salt concentration. The dependence of the process on the pulse amplitude can be utilized to tune the droplet size. To demonstrate the applicability of the electric-field-driven droplet generator, it is shown that the droplets can be used as versatile biological reaction compartments. It is proven that droplets containing a cell-free transcription-translation system execute gene transcription and protein biosynthesis in a timely and programmable fashion. Moreover, it is verified that biomolecules inside the aqueous droplets such as small RNAs can be diffusionally activated from the outside to induce a ligand-driven biochemical switch.

A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System.

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 one-pot method makes the PURE system easy to generate and allows it to be readily optimized and modified.

An educational module to explore CRISPR technologies with a cell-free transcription-translation system.

Within the last 6 years, CRISPR-Cas systems have transitioned from adaptive defense systems in bacteria and archaea to revolutionary genome-editing tools. The resulting CRISPR technologies have driven innovations for treating genetic diseases and eradicating human pests while raising societal questions about gene editing in human germline cells as well as crop plants. Bringing CRISPR into the classroom therefore offers a means to expose students to cutting edge technologies and to promote discussions about ethical questions at the intersection of science and society. However, working with these technologies in a classroom setting has been difficult because typical experiments rely on cellular systems such as bacteria or mammalian cells. We recently reported the use of an E. coli cell-free transcription-translation (TXTL) system that simplifies the demonstration and testing of CRISPR technologies with shorter experiments and limited equipment. Here, we describe three educational modules intended to expose undergraduate students to CRISPR technologies using TXTL. The three sequential modules comprise (i) designing the RNAs that guide DNA targeting, (ii) measuring DNA cleavage activity in TXTL and (iii) testing how mutations to the targeting sequence or RNA backbone impact DNA binding and cleavage. The modules include detailed protocols, questions for group discussions or individual evaluation, and lecture slides to introduce CRISPR and TXTL. We expect these modules to allow students to experience the power and promise of CRISPR technologies in the classroom and to engage with their instructor and peers about the opportunities and potential risks for society.

The Francisella novicida Cas12a is sensitive to the structure downstream of the terminal repeat in CRISPR arrays

ABSTRACTThe Class 2 Type V-A CRISPR effector protein Cas12a/Cpf1 has gained widespread attention in part because of the ease in achieving multiplexed genome editing, gene regulation, and DNA detection. Multiplexing derives from the ability of Cas12a alone to generate multiple guide RNAs from a transcribed CRISPR array encoding alternating conserved repeats and targeting spacers. While array design has focused on how to optimize guide-RNA sequences, little attention has been paid to sequences outside of the CRISPR array. Here, we show that a structured hairpin located immediately downstream of the 3ʹ repeat interferes with utilization of the adjacent encoded guide RNA by Francisella novicida (Fn)Cas12a. We first observed that a synthetic Rho-independent terminator immediately downstream of an array impaired DNA cleavage based on plasmid clearance in E. coli and DNA cleavage in a cell-free transcription-translation (TXTL) system. TXTL-based cleavage assays further revealed that inhibition was associated with incomplete processing of the transcribed CRISPR array and could be attributed to the stable hairpin formed by the terminator. We also found that the inhibitory effect partially extended to upstream spacers in a multi-spacer array. Finally, we found that removing the terminal repeat from the array increased the inhibitory effect, while replacing this repeat with an unprocessable terminal repeat from a native FnCas12a array restored cleavage activity directed by the adjacent encoded guide RNA. Our study thus revealed that sequences surrounding a CRISPR array can interfere with the function of a CRISPR nuclease, with implications for the design and evolution of CRISPR arrays.