Measurement and Numerical Modeling of Cell-Free Protein Synthesis: Combinatorial Block-Variants of the PURE System

Protein synthesis is at the core of bottom-up construction of artificial cellular mimics. Intriguingly, several reports have revealed that when a transcription–translation (TX–TL) kit is encapsulated inside lipid vesicles (or water-in-oil droplets), high between-vesicles diversity is observed in terms of protein synthesis rate and yield. Stochastic solute partition can be a major determinant of these observations. In order to verify that the variation of TX–TL components concentration brings about a variation of produced protein rate and yield, here we directly measure the performances of the 'PURE system' TX–TL kit variants. We report and share the kinetic traces of the enhanced Green Fluorescent Protein (eGFP) synthesis in bulk aqueous phase, for 27 combinatorial block-variants. The eGFP production is a sensitive function of TX–TL components concentration in the explored concentration range. Providing direct evidence that protein synthesis yield and rate actually mirror the TX–TL composition, this study supports the above-mentioned hypothesis on stochastic solute partition, without excluding, however, the contribution of other factors (e.g., inactivation of components)

Extrinsic stochastic factors (solute partition) in gene expression inside lipid vesicles and lipid-stabilized water-in-oil droplets: a review

Abstract The encapsulation of transcription–translation (TX–TL) machinery inside lipid vesicles and water-in-oil droplets leads to the construction of cytomimetic systems (often called 'synthetic cells') for synthetic biology and origins-of-life research. A number of recent reports have shown that protein synthesis inside these microcompartments is highly diverse in terms of rate and amount of synthesized protein. Here, we discuss the role of extrinsic stochastic effects (i.e. solute partition phenomena) as relevant factors contributing to this pattern. We evidence and discuss cases where between-compartment diversity seems to exceed the expected theoretical values. The need of accurate determination of solute content inside individual vesicles or droplets is emphasized, aiming at validating or rejecting the predictions calculated from the standard fluctuations theory. At the same time, we promote the integration of experiments and stochastic modeling to reveal the details of solute encapsulation and intra-compartment reactions

Synthetic in vitro transcriptional oscillators

The construction of synthetic biochemical circuits from simple components illuminates how complex behaviors can arise in chemistry and builds a foundation for future biological technologies. A simplified analog of genetic regulatory networks, in vitro transcriptional circuits, provides a modular platform for the systematic construction of arbitrary circuits and requires only two essential enzymes, bacteriophage T7 RNA polymerase and Escherichia coli ribonuclease H, to produce and degrade RNA signals. In this study, we design and experimentally demonstrate three transcriptional oscillators in vitro. First, a negative feedback oscillator comprising two switches, regulated by excitatory and inhibitory RNA signals, showed up to five complete cycles. To demonstrate modularity and to explore the design space further, a positive-feedback loop was added that modulates and extends the oscillatory regime. Finally, a three-switch ring oscillator was constructed and analyzed. Mathematical modeling guided the design process, identified experimental conditions likely to yield oscillations, and explained the system's robust response to interference by short degradation products. Synthetic transcriptional oscillators could prove valuable for systematic exploration of biochemical circuit design principles and for controlling nanoscale devices and orchestrating processes within artificial cells

Bottom-up construction of complex biomolecular systems with cell-free synthetic biology

Cell-free systems offer a promising approach to engineer biology since their open nature allows for well-controlled and characterized reaction conditions. In this review, we discuss the history and recent developments in engineering recombinant and crude extract systems, as well as breakthroughs in enabling technologies, that have facilitated increased throughput, compartmentalization, and spatial control of cell-free protein synthesis reactions. Combined with a deeper understanding of the cell-free systems themselves, these advances improve our ability to address a range of scientific questions. By mastering control of the cell-free platform, we will be in a position to construct increasingly complex biomolecular systems, and approach natural biological complexity in a bottom-up manner

Steps toward cell-like systems: spatio-temporal control of shared molecular resources for cell-free gene expression

The biosphere offers many promising economically and environmentally sustainable solutions to humanity’s increasing energy demand such as biomass conversion, chemical production, and pharmaceutical fermentation. These solutions could close the carbon loop and improve manufacturing efficiencies, but they all depend on accurate control of protein expression. To avoid limitations to protein control present in vivo such as membranes, homeostasis, and growth, artificial cell-like systems are being researched. These simplified systems are currently useful to study individual aspects of life such as regulating energy flux across membranes, responding to the environment, replication, and growth. These systems could be made more complex in the future to provide a simplified, engineered, cell-like platform for bioprocessing. Even at the single gene level, control of protein expression is hindered by resource sharing and bursting. To make proteins, genes require many reusable resources such as polymerase, ribosomes, and tRNAs which are shared among different genes. Resource sharing causes correlations in protein populations and limits steady state concentrations of competing genes. Bursty gene expression, periods of high expression, and thus high resource use, separated by periods of no expression, and thus no resource use, is a ubiquitous biological phenomenon that intimately links expression bursting and resource sharing. This dissertation investigates how gene expression bursting and variation is affected by expression resources being shared among genes and how the location of expression resources, either encapsulated or outside permeable lipid membranes, controls the level and the dynamics of cell-free protein expression. Cell-free protein synthesis systems, both crude and PURE, areused in combination with both physical PDMS barriers and defined lipid membranes to study the effects of shared and divided resource pools on gene expression bursting and protein production. Experimental results are supplemented with Gillespie simulations to add further insights. This work provides fundamental knowledge of protein expression and applied knowledge of the effects of resource sharing on cell-free gene expression bursting and variation in protein expression confined to cell-relevant volumes which are important steps toward artificial cell-like systems

A Role for Bottom-Up Synthetic Cells in the Internet of Bio-Nano Things?

he potential role of bottom-up Synthetic Cells (SCs) in the Internet of Bio-Nano Things (IoBNT) is discussed. In particular, this perspective paper focuses on the growing interest in networks of biological and/or artificial objects at the micro- and nanoscale (cells and subcellular parts, microelectrodes, microvessels, etc.), whereby communication takes place in an unconventional manner, i.e., via chemical signaling. The resulting “molecular communication” (MC) scenario paves the way to the development of innovative technologies that have the potential to impact biotechnology, nanomedicine, and related fields. The scenario that relies on the interconnection of natural and artificial entities is briefly introduced, highlighting how Synthetic Biology (SB) plays a central role. SB allows the construction of various types of SCs that can be designed, tailored, and programmed according to specific predefined requirements. In particular, “bottom-up” SCs are briefly described by commenting on the principles of their design and fabrication and their features (in particular, the capacity to exchange chemicals with other SCs or with natural biological cells). Although bottom-up SCs still have low complexity and thus basic functionalities, here, we introduce their potential role in the IoBNT. This perspective paper aims to stimulate interest in and discussion on the presented topics. The article also includes commentaries on MC, semantic information, minimal cognition, wetware neuromorphic engineering, and chemical social robotics, with the specific potential they can bring to the IoBNT

A Role for Bottom-Up Synthetic Cells in the Internet of Bio-Nano Things?

The potential role of bottom-up Synthetic Cells (SCs) in the Internet of Bio-Nano Things (IoBNT) is discussed. In particular, this perspective paper focuses on the growing interest in networks of biological and/or artificial objects at the micro- and nanoscale (cells and subcellular parts, microelectrodes, microvessels, etc.), whereby communication takes place in an unconventional manner, i.e., via chemical signaling. The resulting "molecular communication" (MC) scenario paves the way to the development of innovative technologies that have the potential to impact biotechnology, nanomedicine, and related fields. The scenario that relies on the interconnection of natural and artificial entities is briefly introduced, highlighting how Synthetic Biology (SB) plays a central role. SB allows the construction of various types of SCs that can be designed, tailored, and programmed according to specific predefined requirements. In particular, "bottom-up" SCs are briefly described by commenting on the principles of their design and fabrication and their features (in particular, the capacity to exchange chemicals with other SCs or with natural biological cells). Although bottom-up SCs still have low complexity and thus basic functionalities, here, we introduce their potential role in the IoBNT. This perspective paper aims to stimulate interest in and discussion on the presented topics. The article also includes commentaries on MC, semantic information, minimal cognition, wetware neuromorphic engineering, and chemical social robotics, with the specific potential they can bring to the IoBNT

Systems biochemistry of macromolecular interactions involved in the regulation of the division ring stability in bacteria

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, leída el 14-12-2022En Escherichia coli la división se encuentra mediada por el divisoma, un anillo contráctil consistente en un complejo multiproteico posicionado con gran precisión en el centro de la célula (correspondiente al punto medio del eje largo de este bacilo) hacia el final del ciclo celular. El primer paso en la formación de esta maquinaria de división es el ensamblaje del denominado anillo Z, una estructura macromolecular dinámica que implica la polimerización de la proteína conservada FtsZ y que actúa como plataforma sobre la que se incorporan el resto de proteínas de división. El correcto posicionamiento espacial y temporal del anillo Z es crítico para la generación de células hijas viables, dado que desviaciones en la adecuada localización del mismo pueden conllevar consecuencias catastróficas para la bacteria como la bisección del cromosoma o la generación de células anucleadas. Por este motivo, E. coli cuenta con mecanismos de posicionamiento que aseguran la formación del anillo en el centro celular, de entre los cuales el sistema Min y el sistema de oclusión por nucleoide son los más estudiados. El sistema Min establece un patrón de oscilaciones que impide la formación del anillo en los polos celulares a la vez que permite su ensamblaje en el centro de la célula. Por su parte, el sistema de oclusión por nucleoide inhibe la formación del anillo Z en las proximidades del cromosoma bacteriano a través de la interacción directa de la proteína SlmA, unida a secuencias específicas de ADN (SBS) en el cromosoma, con FtsZ. Además, la acción conjunta de estos dos mecanismos reguladores negativos está complementada por el anclaje Ter, consistente en una red multiproteica en la que participan las proteínas ZapA, ZapB y la proteína de unión a ADN MatP que refuerza la formación del anillo Z en posiciones centrales...Escherichia coli cell division is mediated by the divisome, a contractile ring consisting of a multiprotein complex that accurately assembles at midcell (corresponding to the long axis midpoint of this bacillus) toward the end of the cell cycle. The first step in the formation of this division machinery is the assembly of the so-called Z-ring, a dynamic macromolecular structure involving the polymerization of the conserved protein FtsZ that serves as a scaffold for the recruitment of the rest of division proteins. Correct spatiotemporal assembly of theZ-ring is critical for the generation of viable daughter cells, since deviations in its positioning might result in catastrophic outcomes such as chromosome bisection or generation of anucleate cells. For this reason, E. coli relies on dedicated positioning mechanisms to ensure proper formation of the Z-ring at midcell, being the canonical ones the Min system and nucleoid occlusion, which act by blocking the assembly of the Z-ring at undesired locations. The Min system establishes an oscillation pattern that prevents Z-ring formation at the cell poles while allowing its assembly in the cell center. For its part, nucleoid occlusion inhibits Z-ring assembly in the vicinity of the chromosome through direct interaction of the Slm Aprotein, bound to specific DNA sequences (SBS) in the chromosome, with FtsZ.Besides, the concerted action of these two negative regulatory mechanisms is complemented by the Ter linkage, a protein network comprising ZapA, ZapB and the DNA-binding protein MatP that reinforces the formation of the Z-ring at midcell...Fac. de Ciencias QuímicasTRUEunpu