Development of cell-free transcription translation.

Protein degradation is a fundamental process in all living cells and is essential to remove both damaged proteins and intact proteins that are no longer needed by the cell. We are interested in creating synthetic genetic circuits that function in a cell-free expression system. This will require not only an efficient protein expression platform but also a robust protein degradation system in cell extract. Therefore, we purified and tested the activity of E. coli ClpXP protease in cell-free transcription-translation (TX-TL) systems that used E. coli S30 cell extract. Surprisingly, our studies showed that purified ClpXP added to the TX-TL system has very low proteolytic activity. The low activity of ClpXP was correlated with the rapid consumption of adenosine triphosphate (ATP) in cell extract. We improved the activity of ClpXP in cell extract by adding exogenous ATP and an energy regeneration system. We then established conditions for both protein synthesis, and protein degradation by ClpXP to occur simultaneously in the TX-TL systems. The optimized conditions for ClpXP activity will be useful for creating tunable synthetic genetic circuits and in vitro synthetic biology.

The cell-free methodology for the synthesis of functionally active proteins is considered, and the so-called continuous cell-free translation and transcription-translation systems are described. The continuous cell-free systems for gene expression are based on the use of a porous barrier that retains the high-molecular-weight components of the protein-synthesizing machinery within a defined reaction compartment, and at the same time provides the continuous feeding with substrates (NTPs and amino acids) and the removal of reaction products. There are two versions of the continuous systems: the flow version (continuous-flow cell-free, or CFCF systems) and the dialysis version (continuous-exchange cell-free, or CECF systems). Both versions have been shown to provide a prolonged synthesis of proteins, as compared with standard (batch) cell-free systems, and correspondingly a significantly higher yield of proteins synthesized. The synthesis of fusion proteins and the direct expression of PCR products in cell-free systems are discussed as promising methodological approaches in a number of cases. Using the monitoring of polypeptide elongation in cell-free systems the evidence is presented that the folding of synthesized polypeptides into functional protein globules proceeds on ribosomes during translation (co-translational protein folding).

Cell-Free Expression Systems: From Gene Circuits to Self-Assembly Processes in a Test Tube

RNA molecules lie at the heart of living organisms where they are associated with most of the cellular processes. They have recently emerged as one of the most promising elements for developing programmable genetic regulatory systems. RNA regulators have been shown to offer great advantages to harness the power of synthetic biology. Versatility of functions, predictability of design, and light metabolic cost have turned RNA-based devices into components of primordial importance for therapeutic, diagnostic and biotechnological applications. However, advanced tasks require the use of sequential logic circuits that embed many constituents in the same system. Combining RNA-parts into more complex circuits remains experimentally challenging and difficult to predict. Contrary to protein-based networks, little work has been performed regarding the integration of RNA components to multi-level regulated circuits. In the first part of this thesis, combinations of variety of small transcriptional activator RNAs (STARs) and toehold switches were built into highly effective AND-gates. To characterise the components and their dynamic range, an Escherichia coli (E. coli) cell-free transcription-translation (TX-TL) system dispensed via nanoliter droplets was used. Cell-free systems, which constitute an open environment, have removed many of the complexities linked to the traditional use of living cells and have led to exciting opportunities for the rational design of genetic circuits. A modelling framework based on ordinary differential equations (ODEs), where parameters were inferred via parallel tempering, was established to analyse the expression construct in a qualitative and quantitative manner. Based on this analysis, nine additional AND-gates were built and tested 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, displaying a dynamic range comparable to the level of protein circuits. Subsequent spacer screening experiment enabled isolation of a gate mutant with dynamic range up to 1087 fold change, paving the way towards multi-layered devices where tight OFF-stages are required for efficient computation. Expanding the repertoire of RNA regulatory parts with efficient inhibitors would complete the set of logic operations necessary for the building of dynamiccircuits, such as memory devices or oscillators. The TX-TL system was functionalized with pre-expressed dSpyCas9, a mutated version of Cas9 without endonuclease activity. Four functional small guide RNAs (sgRNAs) targeting the sfGFP reporter were engineered and characterized, all resulting in high repression efficiency. A three-inputs logic circuit containing toehold, STAR and sgRNA was successfully co-expressed, validating the orthogonality of NOT and AND gates based solely on RNA-based regulation. In order to minimize interactions which could arise from RNA-circuit of increasing complexity, the TX-TL system was functionalized with a second protein, the Csy4 endoribonuclease, which selectively binds and cuts a small RNA hairpin. Normalization of gene expression from various untranslated region contexts and enhanced processing of three-inputs small RNA operon were demonstrated via the use of Csy4. Finally, characterizing complex RNA-based circuits requires techniques that resolves dynamics. To overcome the batch-format limitations inherent to TX-TL systems, a microfluidic nanoliter-scaled reactor was implemented, enabling synthesis rates to stay constant over time. Dynamic control of RNA circuitry was demonstrated by modulating the concentration of ligands, reversing the gene state through the conformational change of riboswitches. This thesis shows the potential of a rapid prototyping approach for RNA circuit design in TX-TL systems combined with a predicting model framework. Taken together, the characterization of a variety of RNA-parts : activators, repressors, or controllers culminating into logic modules; and augmented cell-extracts; form a complete RNA-toolbox for cell-free systems. The leveraging of this unique prototyping platform will ultimately enable the engineering and the study of highly dynamical RNA-circuits in vitro.

In vitro transcription-translation (TX-TL) can enable faster engineering of biological systems. This speed-up can be significant, especially in difficult-to-transform chassis. This work shows the successful development of TX-TL systems using three soil-derived wild-type Pseudomonads known to promote plant growth: Pseudomonas synxantha, Pseudomonas chlororaphis, and Pseudomonas aureofaciens. All three species demonstrated multiple sonication, runoff, and salt conditions producing detectable protein synthesis. One of these new TX-TL systems, P. synxantha, demonstrated a maximum protein yield of 2.5 μM at 125 proteins per DNA template, a maximum protein synthesis rate of 20 nM/min, and a range of DNA concentrations with a linear correspondence with the resulting protein synthesis. A set of different constitutive promoters driving mNeonGreen expression were tested in TX-TL and integrated into the genome, showing similar normalized strengths for in vivo and in vitro fluorescence. This correspondence between the TX-TL-derived promoter strength and the in vivo promoter strength indicates that these lysate-based cell-free systems can be used to characterize and engineer biological parts without genomic integration, enabling a faster design-build-test cycle.

This work presents the cell-free transcription-translation (TX-TL) system as a research and development platform for renewable synthesis and molecular discovery. TX-TL is easy to use and provides a biomolecular breadboard for the rapid prototyping and engineering of biosynthetic pathways. This work has validated the capabilities of the cell-free TX-TL system for simultaneous protein expression and chemical synthesis. Specifically, this work shows that TX-TL supports the conversion of intermediates from carbohydrate metabolism and amino acids into valuable compounds. Metabolic flux through cofactor dependent pathways confirms that active cofactor metabolism is occurring in TX-TL. This work has also demonstrated the industrial relevance of TX-TL through exploring design space of a biosynthetic pathway for improved product yield and expanding substrate scope of another biosynthetic pathway. Current methods for assembling biosynthetic pathways in microorganisms require a process of repeated trial and error and have long design-build-test cycles. We describe the use of a cell-free transcription-translation (TX-TL) system as a biomolecular breadboard for the rapid engineering of the 1,4-butanediol (BDO) pathway. In this work, we have verified enzyme expression and enzyme activity and identified the conversion of 4-hydroxybutyrate to downstream metabolites as the pathway bottleneck. We demonstrate the reliability of using linear DNA in TX-TL as a tool for engineering biological systems by undertaking a careful characterization of its transcription and translation capabilities and provide a detailed analysis of its metabolic output. Pathway constructs of varying pathway enzyme expression levels are tested in TX-TL and in vivo to identify correlations between the two systems, and we find that the production of BDO is correlated to the expression of enzyme ald in both systems. The use of TX-TL to survey the design space of the BDO pathway enables rapid tuning of pathway enzyme expression levels for improved product yield. Different pathway combinations are also tested in TX-TL for its application in pathway ranking. Leveraging TX-TL to screen enzyme variants for improved catalytic activity accelerates design iterations that can be directly applied to in vivo strain development. TX-TL simulates a customizable cellular environment that can be controlled by manipulating pH, changing cellular components, or adding exogenous substrates. By adding linear DNA encoding individual enzymes of the violacein pathway and tryptophan analogs in TX-TL reactions, we have discovered new violacein analogs. TX-TL enables rapid production of natural product analogs with diverse substitution, which allows small-scale biosynthesis of potential drug candidates and offers a new platform for drug discovery. This work also presents TX-TL as a platform for protein engineering. Residues targeted for site-saturated mutagenesis were identified with protein-ligand docking. Linear DNAs of individual enzyme mutants were added into TX-TL reactions to screen for improved enzyme variant. Screening result indicates vioE mutant Y17H reduces byproduct formation and redirects metabolic flux towards target metabolites. Protein engineering for improved enzyme activity can further expand the substrate scope of a natural product pathway and result with more natural product analogs that can be applied for medical applications. This work demonstrates that the cell-free TX-TL system can become a valuable tool that complements the process of engineering biosynthesis in the whole cell in vivo system or the purified protein in vitro system. Future engineering and development of the TX-TL system can further expand the chemical space for biosynthesis.

TFIID is a multiprotein complex comprised of the TATA-binding protein (TBP) and an array of TBP-associated factors (TAFIIs). Whereas TBP is sufficient for basal transcription in conjunction with other general transcription factors and RNA polymerase II, TAFIIs are additionally required for activator-dependent transcription in mammalian cell-free transcription systems. However, recent in vivo studies carried out in yeast suggest that TAFIIs are not globally required for activator function. The discrepancy between in vivo yeast studies and in vitro mammalian cell-free systems remains to be resolved. In this study, we describe a mammalian cell-free transcription system reconstituted with only recombinant proteins and epitope-tagged multiprotein complexes. Transcriptional activation can be recapitulated in this highly purified in vitro transcription system in the absence of TAFIIs. This TBP-mediated activation is not induced by human mediator, another transcriptional coactivator complex potentially implicated in activator response. In contrast, general transcription factors TFIIH and TFIIA play a significant role in TBP-mediated activation, which can be detected in vitro with Gal4 fusion proteins containing various transcriptional activation domains. Our data, therefore, suggest that TFIIH and TFIIA can mediate activator function in the absence of TAFIIs.

Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as engineering genetic parts, validating genetic circuits, and biosensor outputs. Currently, only two luciferases (Firefly and Renilla) are commonly used without substrate cross-talk. Here we demonstrate the expansion of the cell-free luciferase reporter system, with two orthogonal luciferase reporters: N. nambi luciferase (Luz) and LuxAB. These luciferases do not have cross-reactivity with the Firefly and Renilla substrates. We also demonstrate a substrate regeneration pathway for one of the new luciferases, enabling long-term time courses of protein expression monitoring in the cell-free system. Furthermore, we reduced the number of genes required in TXTL expression, by engineering a cell extract containing part of the luciferase enzymes. Our findings lead to an expanded platform with multiple orthogonal luminescence translation readouts for in vitro protein expression.

Native cell-free transcription-translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription-translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription-translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications.

Cell-free transcription-translation (TXTL) systems expressing genes from linear dsDNA enable the rapid prototyping of genetic devices while avoiding cloning steps. However, repetitive inclusion of a reporter gene is an incompressible cost and sometimes accounts for most of the synthesized DNA length. Here we present reporter systems based on split-GFP systems that reassemble into functional fluorescent proteins and can be used to monitor gene expression in E. coli TXTL. The 135 bp GFP10-11 fragment produces a fluorescent signal comparable to its full-length GFP counterpart when reassembling with its complementary protein synthesized from the 535 bp fragment expressed in TXTL. We show that split reporters can be used to characterize promoter libraries, with data qualitatively comparable to full-length GFP and matching in vivo expression measurements. We also use split reporters as small fusion tags to measure the TXTL protein and peptide production yield. Finally, we generalize our concept by providing a luminescent split reporter based on split-nanoluciferase. The ∼80% gene sequence length reduction afforded by split reporters lowers synthesis costs and liberates space for testing larger devices while producing a reliable output. In the peptide production context, the small size of split reporters compared with full-length GFP is less likely to bias peptide solubility assays. We anticipate that split reporters will facilitate rapid and cost-efficient genetic device prototyping, protein production, and interaction assays.

Chapter 21 - Advances and applications of cell-free systems for metabolic production

Cell-free transcription-translation (TX-TL) systems have been used for diverse applications, but their performance and scope are limited by variability and poor predictability. To understand the drivers of this variability, we explored the effects of metabolic perturbations to anEscherichia coli (E. coli) Rosetta2 TX-TL system. We targeted three classes of molecules: energy molecules, in the form of nucleotide triphosphates (NTPs); central carbon "fuel" molecules, which regenerate NTPs; and magnesium ions (Mg2+). Using malachite green mRNA aptamer (MG aptamer) and destabilized enhanced green fluorescent protein (deGFP) as transcriptional and translational readouts, respectively, we report the presence of a trade-off between optimizing total protein yield and optimizing total mRNA yield, as measured by integrating the area under the curve for mRNA time-course dynamics. We found that a system's position along the trade-off curve is strongly determined by Mg2+ concentration, fuel type and concentration, and cell lysate preparation and that variability can be reduced by modulating these components. Our results further suggest that the trade-off arises from limitations in translation regulation and inefficient energy regeneration. This work advances our understanding of the effects of fuel and energy metabolism on TX-TL in cell-free systems and lays a foundation for improving TX-TL performance, lifetime, standardization, and prediction.

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 0.8µM 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.

The bottom-up synthesis of a minimal biological cell is achieved by integrating and connecting three fundamental modules: metabolism, information and self-organization (Noireaux et al., 2011). The execution and connection of these three parts into cell-sized liposomes should generate a system capable of self-reproduction and ultimately evolution. Each of these molecular sub systems is the result of a forward engineering process where bio-parts, e.g. sugars, proteins, phospholipids and nucleotides are interlocked in a functional way (Mann 2008). The creation of a minimal biological cell is certainly one of the most challenging goals of the synthetic biology community (Porcar et al., 2011). The objective of this research area is to elucidate the fundamental design principles found in biology and to understand cellular functions by applying a reductionist approach (Elowitz, 2010). This work also promotes the development of new technologies based on life’s principles (Bedau et al. 2010). The functionalities of biological cells are dependent on the activity of molecular machineries (enzymes). However, such machineries require energy for their physiological function. Therefore, in order to attempt the construction of wet artificial life is essential to reconstitute an in vitro metabolic network that supports the cellular energetic. In biological systems, the energy requirements are stored in the high-energy molecule adenosine triphosphate (ATP). The conversion from ATP to adenosine diphosphate (ADP) is crucial for energy generation. In particular, the hydrolysis of one chemical bond releases inorganic phosphate (iP) and liberates energy used for life processes. Noireaux’s lab has recently developed a unique cell-free transcription-translation (TX-TL) system for synthetic biology applications, as for instance, the possibility of executing DNA programs made of up to ≈ 60 genes (Shin and Noireaux, 2012a, 2012b). In a cell-free system, the rate of protein synthesis depends exponentially on the adenylate energy charge (Atkinson 1968; Matveev et al., 1996). Protein degradation by AAA+ proteases is also highly dependent on the pool of chemical energy available. Therefore, efficient long-lived ATP regeneration and byproducts recycling is at the heart of a minimal cell construction by allowing the execution of larger and larger DNA programs with interesting dynamical behaviors. We will present our recent efforts to design an in vitro metabolism for efficient protein synthesis. This new metabolic scheme relies on the catabolism of polysugars molecules as energetic resources, and it only exploits the endogenous enzymes present in the cellular extract. Cell-free protein synthesis is improved by addition of maltose or maltodextrin in the reaction mixture. The initial phosphorylation of maltose or maltodextrin produces either glucose or glucose-6-phosphate, which are intermediates of the glycolysis. In turn, this allows for higher level of sustained ATP concentration through recycling of iP, the byproduct of the transcription/translation processes. We will present biochemical experiments that quantitatively measure several system’s parameters: concentration of synthesized protein (a reporter gene eGFP), level of ATP and inorganic phosphate, as well as pH fluctuations during in vitro protein synthesis. Recently, we reported the highest protein yield ever achieved with an E. coli cell-free expression system with this new metabolism (Caschera and Noireaux 2013). Therefore, compared to others cell-free expression systems used to design a minimal cell (Ichihashi et al. 2010), it represents a more powerful solution in term of adenylate energy charge. We are now using this system to develop a minimal cell. One of the current bottlenecks in this research area is the encapsulation of the cell-free TX-TL reaction into cell-sized vesicles of complex phospholipid composition. Further optimization of the reaction mixture for cell-free protein synthesis, as well as its integration into liposome with an active membrane (Noireaux and Libchaber 2004), could be accelerated exploiting a machine learning approach coupled to robotic workstation for liquid handling (Caschera et al. 2010, 2011).

Towards the assembly of a minimal oscillator