Cell-free synthesis of the Salmonella specific broad host range bacteriophage, felixO1.

Phage-based biocontrol of foodborne Salmonella is limited by the requisite use of Salmonella to propagate the phages. This limitation can be circumvented by producing Salmonella phages using a cell-free gene expression system (CFE) with a non-pathogenic chassis. Here, we produce the Salmonella phage felixO1 using an E. coli-based CFE system.

The all-E. coliTXTL toolbox 3.0: new capabilities of a cell-free synthetic biology platform.

The new generation of cell-free gene expression systems enables the prototyping and engineering of biological systems in vitro over a remarkable scope of applications and physical scales. As the utilization of DNA-directed in vitro protein synthesis expands in scope, developing more powerful cell-free transcription-translation (TXTL) platforms remains a major goal to either execute larger DNA programs or improve cell-free biomanufacturing capabilities. In this work, we report the capabilities of the all-E. coli TXTL toolbox 3.0, a multipurpose cell-free expression system specifically developed for synthetic biology. In non-fed batch-mode reactions, the synthesis of the fluorescent reporter protein eGFP (enhanced green fluorescent protein) reaches 4 mg/ml. In synthetic cells, consisting of liposomes loaded with a TXTL reaction, eGFP is produced at concentrations of >8 mg/ml when the chemical building blocks feeding the reaction diffuse through membrane channels to facilitate exchanges with the outer solution. The bacteriophage T7, encoded by a genome of 40 kb and ∼60 genes, is produced at a concentration of 1013 PFU/ml (plaque forming unit/ml). This TXTL system extends the current cell-free expression capabilities by offering unique strength and properties, for testing regulatory elements and circuits, biomanufacturing biologics or building synthetic cells.

Phase Separation and Protein Partitioning in Compartmentalized Cell-Free Expression Reactions.

Liquid-liquid phase separation (LLPS) is important to control a wide range of reactions from gene expression to protein degradation in a cell-sized space. To bring a better understanding of the compatibility of such phase-separated structures with protein synthesis, we study emergent LLPS in a cell-free transcription-translation (TXTL) reaction. When the TXTL reaction composed of many proteins is concentrated, the uniformly mixed state becomes unstable, and membrane-less phases form spontaneously. This LLPS droplet formation is induced when the TXTL reaction is enclosed in water-in-oil emulsion droplets, in which water evaporates from the surface. As the emulsion droplets shrink, smaller LLPS droplets appear inside the emulsion droplets and coalesce into large phase-separated domains that partition the localization of synthesized reporter proteins. The presence of PEG in the TXTL reaction is important not only for versatile cell-free protein synthesis but also for the formation of two large domains capable of protein partitioning. Our results may shed light on the dynamic interplay of LLPS formation and cell-free protein synthesis toward the construction of synthetic organelles.

Complex dependence of CRISPR-Cas9 binding strength on guide RNA spacer lengths.

It is established that for CRISPR-Cas9 applications guide RNAs with 17-20 bp long spacer sequences are optimal for accurate target binding and cleavage. In this work we perform cell-free CRISPRa (CRISPR activation) and CRISPRi (CRISPR inhibition) experiments to demonstrate the existence of a complex dependence of CRISPR-Cas9 binding as a function of the spacer length and complementarity. Our results show that significantly truncated or mismatched spacer sequences can form stronger guide-target bonds than the conventional 17-20 bp long spacers. To explain this phenomenon, we take into consideration previous structural and single-molecule CRISPR-Cas9 experiments and develop a novel thermodynamic model of CRISPR-Cas9 target recognition.

Programming multi-protein assembly by gene-brush patterns and two-dimensional compartment geometry.

The assembly of protein machines in cells is precise, rapid, and coupled to protein synthesis with regulation in space and time. The assembly of natural and synthetic nanomachines could be similarly controlled by genetic programming outside the cell. Here, we present quasi-two-dimensional (2D) silicon compartments that enable programming of protein assembly lines by local synthesis from surface-immobilized DNA brushes. Using this platform, we studied the autonomous synthesis and assembly of a structural complex from a bacteriophage and a bacterial RNA-synthesizing machine. Local synthesis and surface capture of complexes provided high assembly yield and sensitive detection of spatially resolved assembly intermediates, with the 3D geometry of the compartment and the 2D pattern of brushes dictating the yield and mode of assembly steps. Localized synthesis of proteins in a single gene brush enhances their interactions, and displacement of their genes in separated brushes leads to step-by-step surface assembly. This methodology enables spatial regulation of protein synthesis, and deciphering, reconstruction and design of biological machine assembly lines.

Analysis of Cytoplasmic and Membrane Molecular Crowding in Genetically Programmed Synthetic Cells.

Building genetically programmed synthetic cell systems by molecular integration is a powerful and effective approach to capture the synergies between biomolecules when they are put together. In this work, we characterized quantitatively the effects of molecular crowding on gene expression in the cytoplasm of minimal cells, when a crowding agent is added to the reaction, and on protein self-assembly at the membrane, when a crowding agent is attached to the lipid bilayer. We demonstrate that achieving membrane crowding only is sufficient to keep cytoplasmic expression at its highest and to promote the polymerization of the MreB cytoskeletal protein at the lipid bilayer into a network that is mechanically sturdy. Furthermore, we show that membrane crowding can be emulated by different types of macromolecules, supporting a purely entropic mode of action for supramolecular assembly of cytoskeletal proteins at the bilayer. These unanticipated results provide quantitative and general insights relevant to synthetic cell builders.

Membrane molecular crowding enhances MreB polymerization to shape synthetic cells from spheres to rods.

Executing gene circuits by cell-free transcription-translation into cell-sized compartments, such as liposomes, is one of the major bottom-up approaches to building minimal cells. The dynamic synthesis and proper self-assembly of macromolecular structures inside liposomes, the cytoskeleton in particular, stands as a central limitation to the development of cell analogs genetically programmed. In this work, we express the Escherichia coli gene mreB inside vesicles with bilayers made of lipid-polyethylene glycol (PEG). We demonstrate that two-dimensional molecular crowding, emulated by the PEG molecules at the lipid bilayer, is enough to promote the polymerization of the protein MreB at the inner membrane into a sturdy cytoskeleton capable of transforming spherical liposomes into elongated shapes, such as rod-like compartments. We quantitatively describe this mechanism with respect to the size of liposomes, lipid composition of the membrane, crowding at the membrane, and strength of MreB synthesis. So far unexplored, molecular crowding at the surface of synthetic cells emerges as an additional development with potential broad applications. The symmetry breaking observed could be an important step toward compartment self-reproduction.

Multiplex transcriptional characterizations across diverse bacterial species using cell-free systems.

Cell-free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell-free measurements is limited by the use of channel-limited fluorescent readouts. Here, we describe DNA Regulatory element Analysis by cell-Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employ this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from > 1,000 diverse regulatory sequences reveals functional differences in promoter activity that can be quantitatively modeled, providing a rich resource for tuning gene expression in diverse bacterial species. Finally, we examine the transcriptional capacities of dual-species hybrid lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell-free multiplex transcriptional measurement approach will improve genetic part prototyping in new bacterial chassis for synthetic biology.

Gene Expression in on-Chip Membrane-Bound Artificial Cells.

Artificial cells made of molecular components and lipid membrane are emerging platforms to characterize living systems properties. Cell-free transcription-translation (TXTL) offers advantages for the bottom-up synthesis of cellular reactors. Yet, scaling up their design within well-defined geometries remains challenging. We present a microfluidic device hosting TXTL reactions of a reporter gene in thousands of microwells separated from an external buffer by a phospholipid membrane. In the presence of nutrients in the buffer, microreactors are stable beyond 24 h and yield a few mg/mL of proteins. Nutrients in the external solution feed the TXTL reaction at the picoliter scale via passive transport across the phospholipid membrane of each microfluidic well, despite the absence of pores. Replacing nutrients with an inert polymer and fatty acids at an isotonic concentration reduces microreactors efficiency, and a significant fraction yields no protein. This emphasizes the crucial role of the membrane for designing cell-free TXTL microreactors as efficient artificial cells.

Characterization of the all-E. coli transcription-translation system myTXTL by mass spectrometry.

RATIONALE: Cell-free transcription-translation (TXTL) is becoming a popular technology to prototype and engineer biological systems outside living organisms. TXTL relies commonly on a cytoplasmic extract that provides the molecular components necessary to recapitulate gene expression in vitro, where most of the available systems are derived from E. coli. The proteinic and enzymatic composition of lysates, however, is typically unknown. In this work, we analyzed by mass spectrometry the molecular constituents of the all-E. coli TXTL platform myTXTL prepared from the E. coli strain BL21 Rosetta2. METHODS: Standard TXTL reactions were assembled and executed for 10-12 hours at 29°C. In addition to a no-DNA control, four DNA programs were executed in separate reactions to synthesize the reporter protein deGFP as well as the phages MS2, phix174 and T7. The reactions were treated according to standard procedures (trypsin treatment, cleaning) before performing liquid chromatography/mass spectrometry (LC/MS). Data analysis was performed using Sequest and protein identification using Scaffold. RESULTS: A total of 500-800 proteins were identified by LC/MS in the blank reactions. We organized the most abundant protein sets into several categories pertaining, in particular, to transcription, translation and ATP regeneration. The synthesis of deGFP was easily measured. The major structural proteins that compose the three phages MS2, phix174 and T7 were also identified. CONCLUSIONS: Mass spectrometry is a practical tool to characterize biochemical solutions as complex as a cell-free TXTL reaction and to determine the presence of synthesized proteins. The data presented demonstrate that the composition of TXTL based on lysates can be used to validate some underlying molecular mechanisms implicated in cell-free protein synthesis. The composition of the lysate shows significant differences with respect to similar studies on other E. coli strains.

TXTL-based approach to synthetic cells.

Cell-free transcription-translation (TXTL) has recently emerged as a versatile technology to engineer biological systems. In this chapter, we show how an all E. coli TXTL system can be used to build synthetic cell prototypes. We describe methods to encapsulate TXTL reactions in cell-sized liposomes, with an emphasis on the composition of the external solution and lipid bilayer. Cell-free expression is quantitatively described in bulk reactions and liposomes for three proteins: the soluble reporter protein eGFP, the membrane proteins alpha-hemolysin (AH) from Staphylococcus aureus, and the mechanosensitive channel of large conductance (MscL) from E. coli.

Cell-free transcription-translation: engineering biology from the nanometer to the millimeter scale.

Cell-free transcription-translation (TXTL) has become a highly versatile technology to construct, characterize and interrogate genetically programmed biomolecular systems implemented outside living organisms. By recapitulating gene expression in vitro, TXTL offers unparalleled flexibility to take apart, engineer and analyze quantitatively the effects of chemical, physical and genetic contexts on the function of biochemical systems, from simple regulatory elements to millimeter-scale pattern formation. Here, we review the capabilities of the current cell-free platforms for executing DNA programs in vitro. We describe the recent advances in programming using cell-free expression, a multidisciplinary playground that has enabled a myriad of novel applications in synthetic biology, biotechnology, and biological physics. Finally, we discuss the challenges and perspectives in the research area of TXTL-based constructive biology.

Sequestration of Proteins by Fatty Acid Coacervates for Their Encapsulation within Vesicles.

Encapsulating biological materials in lipid vesicles is of interest for mimicking cells; however, except in some particular cases, such processes do not occur spontaneously. Herein, we developed a simple and robust method for encapsulating proteins in fatty acid vesicles in high yields. Fatty acid based, membrane-free coacervates spontaneously sequester proteins and can reversibly form membranous vesicles upon varying the pH value, the precrowding feature in coacervates allowing for protein encapsulation within vesicles. We then produced enzyme-enriched vesicles and show that enzymatic reactions can occur in these micrometric capsules. This work could be of interest in the field of synthetic biology for building microreactors.

Clouding in fatty acid dispersions for charge-dependent dye extraction.

The clouding phenomenon in non-ionic surfactant systems is a common feature that remains rare for ionic detergents. Here, we show that fatty acid (negatively charged) systems cloud upon cooling hot dispersions depending on the concentration or when adding excess guanidine hydrochloride. The clouding of these solutions yields the formation of enriched fatty acid droplets in which they exhibit a polymorphism that depends on the temperature: upon cooling, elongated wormlike micelles transit to rigid stacked bilayers inside droplets. Above this transition temperature, droplets coalesce yielding a phase separation between a fatty acid-rich phase and water, allowing extraction of dyes depending on their charge and lipophilicity. Positively charged and zwitterionic dyes were sequestered within the droplets (and then in the fatty acid-rich upper phase) whereas the negatively charged ones were found in both phases. Our results show an additional case of negatively charged surfactant which exhibit clouding phenomenon and suggest that these systems could be used for extracting solutes depending on their charge and lipophilicity.

Bax mitochondrial relocation is linked to its phosphorylation and its interaction with Bcl-xL.

The heterologous expression of Bax, and other Bcl-2 family members, in the yeast Saccharomyces cerevisiae, has proved to be a valuable reporter system to investigate the molecular mechanisms underlying their interaction with mitochondria. By combining the co-expression of Bax and Bcl-xL mutants with analyzes of their localization and interaction in mitochondria and post-mitochondrial supernatants, we showed that the ability of Bax and Bcl-xL to interact is dependent both on Bax phosphorylation - mimicked by a substitution S184D - and by Bax and Bcl-xL localization. This, and previous data, provide the molecular basis for a model of dynamic equilibrium for Bax localization and activation, regulated both by phosphorylation and Bcl-xL.