Chemical communication between synthetic and natural cells: a possible experimental design

The bottom-up construction of synthetic cells is one of the most intriguing and interesting research arenas in synthetic biology. Synthetic cells are built by encapsulating biomolecules inside lipid vesicles (liposomes), allowing the synthesis of one or more functional proteins. Thanks to the in situ synthesized proteins, synthetic cells become able to perform several biomolecular functions, which can be exploited for a large variety of applications. This paves the way to several advanced uses of synthetic cells in basic science and biotechnology, thanks to their versatility, modularity, biocompatibility, and programmability. In the previous WIVACE (2012) we presented the state-of-the-art of semi-synthetic minimal cell (SSMC) technology and introduced, for the first time, the idea of chemical communication between synthetic cells and natural cells. The development of a proper synthetic communication protocol should be seen as a tool for the nascent field of bio/chemical-based Information and Communication Technologies (bio-chem-ICTs) and ultimately aimed at building soft-wet-micro-robots. In this contribution (WIVACE, 2013) we present a blueprint for realizing this project, and show some preliminary experimental results. We firstly discuss how our research goal (based on the natural capabilities of biological systems to manipulate chemical signals) finds a proper place in the current scientific and technological contexts. Then, we shortly comment on the experimental approaches from the viewpoints of (i) synthetic cell construction, and (ii) bioengineering of microorganisms, providing up-to-date results from our laboratory. Finally, we shortly discuss how autopoiesis can be used as a theoretical framework for defining synthetic minimal life, minimal cognition, and as bridge between synthetic biology and artificial intelligence.Comment: In Proceedings Wivace 2013, arXiv:1309.712

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

Defining the Free Energy Landscape for Protein Induced Cell Membrane Curvature

Using methods from computational statistical mechanics, this thesis aims to elucidate the free energy landscape for protein mediated curvature induction in cell membranes. In particular, a mesoscale model of the cell membrane is utilized in this thesis to probe the thermodynamics of several membrane morphological dependent phenomena including membrane tubulation, the formation of endocytic buds, and protein recruitment on cell protrusions. This model allows for the quantification of membrane proteins curvature sensing behavior due to thermal fluctuations, and is able to predict morphologies which form due to membrane proteins cooperative effects. Analysis of the free energy landscape for generation of tubular membrane structures finds correspondence with the thermodynamics of micelle formation in amphiphilic systems. Furthermore, this research is able to quantify differential protein recruitment on protrusive membrane morphologies and inform cell network models of the interplay between membrane tension and curvature inducing protein signaling

A molecular view on the escape of lipoplexed DNA from the endosome

The use of non-viral vectors for in vivo gene therapy could drastically increase safety, whilst reducing the cost of preparing the vectors. A promising approach to non-viral vectors makes use of DNA/cationic liposome complexes (lipoplexes) to deliver the genetic material. Here we use coarse-grained molecular dynamics simulations to investigate the molecular mechanism underlying efficient DNA transfer from lipoplexes. Our computational fusion experiments of lipoplexes with endosomal membrane models show two distinct modes of transfection: parallel and perpendicular. In the parallel fusion pathway, DNA aligns with the membrane surface, showing very quick release of genetic material shortly after the initial fusion pore is formed. The perpendicular pathway also leads to transfection, but release is slower. We further show that the composition and size of the lipoplex, as well as the lipid composition of the endosomal membrane, have a significant impact on fusion efficiency in our models

Sculpting and fusing biomimetic vesicle networks using optical tweezers

Constructing higher-order vesicle assemblies has discipline-spanning potential from responsive soft-matter materials to artificial cell networks in synthetic biology. This potential is ultimately derived from the ability to compartmentalise and order chemical species in space. To unlock such applications, spatial organisation of vesicles in relation to one another must be controlled, and techniques to deliver cargo to compartments developed. Herein, we use optical tweezers to assemble, reconfigure and dismantle networks of cell-sized vesicles that, in different experimental scenarios, we engineer to exhibit several interesting properties. Vesicles are connected through double-bilayer junctions formed via electrostatically controlled adhesion. Chemically distinct vesicles are linked across length scales, from several nanometres to hundreds of micrometres, by axon-like tethers. In the former regime, patterning membranes with proteins and nanoparticles facilitates material exchange between compartments and enables laser-Triggered vesicle merging. This allows us to mix and dilute content, and to initiate protein expression by delivering biomolecular reaction components

Cell-Free Synthetic Biology Platform for Engineering Synthetic Biological Circuits and Systems

Synthetic biology brings engineering disciplines to create novel biological systems for biomedical and technological applications. The substantial growth of the synthetic biology field in the past decade is poised to transform biotechnology and medicine. To streamline design processes and facilitate debugging of complex synthetic circuits, cell-free synthetic biology approaches has reached broad research communities both in academia and industry. By recapitulating gene expression systems in vitro, cell-free expression systems offer flexibility to explore beyond the confines of living cells and allow networking of synthetic and natural systems. Here, we review the capabilities of the current cell-free platforms, focusing on nucleic acid-based molecular programs and circuit construction. We survey the recent developments including cell-free transcription– translation platforms, DNA nanostructures and circuits, and novel classes of riboregulators. The links to mathematical models and the prospects of cell-free synthetic biology platforms will also be discussed.11Yscopu

Estudios computacionales de mecanismos moleculares de la inmunidad innata

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, leída el 20-12-2022Antimicrobial Resistance (AMR) is a worldwide health emergency. ESKAPE pathogens include the most relevant AMR bacterial families. In particular, Gram-negative bacteria stand out due to their cell envelope complexity, which exhibits strong resistance to antimicrobials. A key element for AMR is the chemical structure of bacterial lipopolysaccharide (LPS), and the phospholipid composition of the membrane, inflecting the membrane permeability to antibiotics. We have applied coarse-grained molecular dynamics simulations to capture the role of the phospholipid composition and lipid A structure in the membrane properties and morphology of ESKAPE Gram-negative bacterial vesicles. Moreover, the reported antimicrobial peptides Cecropin B1, JB95, and PTCDA1-kf were used to unveil their implications for membrane disruption. This study opens a promising starting point for understanding the molecular keys of bacterial membranes and promoting the discovery of new antimicrobials to overcome AMR...La resistencia a los antimicrobianos (AMR) es una emergencia sanitaria mundial. Los patógenos ESKAPE incluyen las familias bacterianas más resistentes a antibióticos y son altamente virulentas. En particular, las bacterias Gram negativas destacan por la complejidad de su pared celular, que presenta una fuerte resistencia frente a los antibióticos. Un elemento clave para la AMR es la estructura química del lipopolisacárido bacteriano (LPS) y la composición de los fosfolípidos de la membrana bacteriana, que influyen en su permeabilidad a los antibióticos. Se han empleado simulaciones de dinámica molecular de grano grueso para captar el papel de la composición de los fosfolípidos y la estructura del LPS en las propiedades y morfología de modelos de vesículas bacterianas Gram negativas ESKAPE. Además, se han empleado los péptidos antimicrobianos Cecropin B1, JB95 y PTCDA1-kf para desvelar su mecanismo disrupción de la membrana bacteriana. Este estudio abre un prometedor punto de partida para comprender las claves moleculares de la resistencia en membranas bacterianas y acelerar el descubrimiento de nuevos antibióticos para hacer frente a la AMR...Fac. de FarmaciaTRUEunpu