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Biology subjects

Tang, T.-Y. D.

Publications and source records attributed to Tang, T.-Y. D..

11 recordsLinked to original sources

Gene expression noise is reduced in communicating synthetic cell populations

A major goal in bottom-up synthetic biology is the construction of multicellular synthetic systems capable of coordinated and robust collective behaviours. However, robustness is often limited by noise and variability arising from increased molecular complexity. Whilst communication has been implemented in synthetic multi-cellular systems, the ability for communication to suppress cell free gene expression variability in populations of synthetic cells remain unexplored. To address this, we encapsulated the Lux and Las quorum sensing gene circuits in lipid vesicles under cell-free conditions to test the effect of communication on reducing cell-free gene expression variability across the population. Our results show that communication, limiting expression resources, and membrane surface effects can reduce gene expression variability. Resource limited Gillespie simulations for transcription and translation show that communication-mediated coupling reduces population-level expression noise under constrained and excess resource conditions. Together, our work provides simple strategies to reduce gene expression variability and thereby improve robustness in synthetic multicellular systems, an important criteria for the future applications of synthetic cells.

synthetic biology↗

Enzyme-driven phase separation of synthetic condensates enables self-organizing compartments and protective microenvironments

Compartmentalization via phase separation is increasingly recognized as a dynamic regulator of cellular biochemistry, yet the consequences of coupling enzymatic reactions to compartmentalization remain poorly understood. We combine a minimal, cell-free expression system with kinetic modeling to show how coupling enzymatic activity to biomolecular condensates drives emergent self-regulation via droplet formation and dissolution. In-vitro mRNA transcription induces phase separation with an intrinsically disordered protein (mutant G3BP1), forming condensates that modulate transcription and degradation kinetics. Our kinetic model shows that phase separation reduces rate constants, with slower degradation within condensates than in the mRNA-protein-poor phase. This leads to more mRNA with a prolonged lifetime of mRNA relative to the case without condensates. Extending the model to sustained and oscillatory resource supply reveals that condensates elevate mean mRNA levels and buffer deviations from the mean compared to the non-condensate scenario. These findings provide a general mechanism of cross-regulation and feedback between phase separation and enzymatic networks, highlighting condensates as active regulators of biochemical flux rather than as passive organizers. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/696465v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d48750org.highwire.dtl.DTLVardef@ffc9eeorg.highwire.dtl.DTLVardef@1222de6org.highwire.dtl.DTLVardef@f36701_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

Absolute Quantification of Fluorescent Protein Fusions by Proteomics

Fusions with fluorescence proteins (FPs) play a pivotal role in experimental biology because of their sensitive and spatially precise visualization by spectroscopy. However, observed fluorescence is not always proportional to their molar abundance. Only a fraction of the fusion protein containing the mature fluorescence chromophore is detectable by spectroscopy and there is no generic method for estimating its molar abundance. We developed a fluorescence-independent mass-spectrometry based approach for accurate absolute sub-femtomole quantification of FP-fusions that also estimates the protein faction having fully matured chromophore. The method exploits an isotopically labelled 68 kDa recombinant protein standard containing peptide proxies for 6 prototypical FPs (mCherry, mScarlet-I, mKate2, EGFP, mNeonGreen and Dendra2) and two self-labelling (Halo- and SNAP-) tags. It enables quantification of proteins fused to any of more than 70 FPs or self-labelling tags. It is versatile, robust, precise and can be used broadly for the absolute quantification of fluorescent fusions in in-vivo and in-vitro cellular systems. As case study we combined mass spectrometry with fluorescence spectroscopy to study expression kinetics of FP fusions in cell-free systems. Molar concentrations of the expressed fusion protein, its fraction containing the mature fluorescent chromophore and of the fluorescent protein were integrated into a mathematical model to obtain kinetic rates of translation, chromophore maturation and folding.

biochemistry↗

Protometabolically generated NADH mediates material properties of aqueous dispersions to coacervate microdroplets

Macromolecular assembly between biomolecules dictates the material state of aqueous dispersions like the cytoplasm. The formation of protein precipitates, fibres, or liquid droplets have been associated with the regulation of biochemical function and disease. However, the effect of small molecules and metabolites on tuning phase behaviour remains underexplored. Here we use the protometabolic reduction of NAD+ to NADH by pyruvate to study the effect of NADH production on the phase properties of polyarginine in bicarbonate buffer. Our results show that reduction of NAD+ in the presence of polyarginine can tune the material properties of the dispersion between precipitates, homogeneous solution and liquid droplets depending on the buffer concentration. In-situ droplet formation results in 2-3 times higher reaction rate and NADH yield, compared to homogeneous solution. Our results demonstrate how small molecules can significantly impact material properties in aqueous dispersions and the positive effect of heterogeneous dispersions on primitive metabolic reactions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/642830v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@19261c8org.highwire.dtl.DTLVardef@151aae5org.highwire.dtl.DTLVardef@aaa110org.highwire.dtl.DTLVardef@342e16_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

Cell free expression in proteinosomes prepared from native protein-PNIPAAm conjugates

Towards the goal of building synthetic cells from the bottom-up, the establishment of micrometer-sized compartments that contain and support cell free transcription and translation that couple cellular structure to function is of critical importance. Proteinosomes, formed from crosslinked cationized protein-polymer conjugates offer a promising solution to membrane-bound compartmentalisation with an open, semi-permeable membrane. Critically, to date, there have been no demonstration of cell free transcription and translation within water-in-water proteinosomes. Herein, we present a novel approach to the fabrication of proteinosomes directly from native protein-polymer (BSA-PNIPAAm) conjugates. We show that these native proteinosomes offer an excellent alternative as artificial cell chassis. Significantly, the native proteinosomes are stable under high salt conditions and can consequently support cell free transcription and translation. The native proteinosomes offer enhanced protein expression compared to proteinosomes prepared from traditional methodologies. Furthermore, we demonstrate the integration of proteinosomes into higher order cellular architectures with membrane free compartments and liposomes. The integration of bioinspired architectural elements with the central dogma is an essential building block for realizing minimal synthetic cells and is key for exploiting artificial cells in real-world applications.

synthetic biology↗

Cell-free quorum sensing to drive feedback and communication within populations of droplet interface bilayers

Building synthetic multicellular systems using non-living molecular components is a grand challenge in the field of bottom-up synthetic biology. Towards this goal, a diverse range of chemistries have been developed to provide mechanisms of intercellular communication and methods to assemble multicellular compartments. However, building bottom-up synthetic multicellular systems is still challenging because it requires the integration of intercellular reaction networks with compatible cellular compartment properties. In this study, we encapsulated cell-free expression systems (CFES) expressing two quorum sensing genetic circuits into droplet interface bilayer (DIB) synthetic cells to demonstrate intercellular communication and feedback. We further develop a method of generating custom DIB multicellular structures by acoustic liquid handling to automatically dispense the CFES droplets and show the potential for multiplexing compartmentalized gene circuits for generating heterogeneous populations of cells. Our work provides a step towards building more complex multicellular systems with feedback mechanisms from the bottom-up to study and experimentally model biological multiscalar processes.

synthetic biology↗

Non-equilibrium conditions inside rock pores drive fission, maintenance and selection of coacervate protocells

Key requirements for the first cells on Earth include the ability to compartmentalize and evolve. Compartmentalization spatially localizes biomolecules from a dilute pool and an evolving cell which grows and divides permits mixing and propagation of information to daughter cells. Complex coacervate microdroplets are excellent candidates as primordial cells with the ability to partition and concentrate molecules into their core and support primitive and complex biochemical reactions. However, the evolution of coacervate protocells by fusion, growth and fission has not yet been demonstrated. In this work, a primordial environment initiated the evolution of coacervate-based protocells. Gas bubbles inside heated rock pores perturb the coacervate protocell distribution and drive the growth, fusion, division and selection of coacervate microdroplets. This setting provides a primordial non-equilibrium environment. Our findings describe how common gas bubbles within heated rock pores induce the early evolution processes of coacervate-based protocells, providing a compelling scenario for the evolution of membrane-free coacervate microdroplets on the early Earth.

biophysics↗

Quantitative Theory for the Diffusive Dynamics of Liquid Condensates

To unravel the biological functions of membraneless liquid condensates it is crucial to develop a quantitative understanding of the physics underlying their dynamics. Key processes within such condensates are diffusion and material exchange with their environment. Experimentally, diffusive dynamics are typically probed via fluorescent labels. However, to date we lack a physics-based quantitative framework for the dynamics of labeled condensate components. Here, we derive the corresponding theory, building on the physics of phase separation, and quantitatively validate this framework via experiments. We show that using our theory we can precisely determine diffusion coefficients inside liquid condensates via a spatio-temporal analysis of fluorescence recovery after photobleaching (FRAP) experiments. We showcase the accuracy and precision of our approach by considering space- and time-resolved data of protein condensates and two different polyelectrolyte-coacervate systems. Strikingly, our theory can also be used to determine the diffusion coefficient in the dilute phase and the partition coefficient, without relying on fluorescence measurements in the dilute phase. This bypasses recently described quenching artefacts in the dense phase, which can underestimate partition coefficients by orders of magnitude. Our experimentally verified theory opens new avenues for theoretically describing molecule dynamics in condensates, measuring concentrations based on the dynamics of fluorescence intensities and quantifying rates of biochemical reactions in liquid condensates.

biophysics↗

Characterization of RNA content in individual phase-separated coacervate microdroplets

Liquid-liquid phase separation or condensation is a form of macromolecular compartmentalization. Condensates formed by complex coacervation were hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of condensation in cells and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and how single condensates differ in their RNA composition. Therefore, we developed an approach to read the RNA content from single condensates using high-throughput sequencing. We find that RNAs which are enriched for specific sequence motifs efficiently accumulate in condensates. These motifs show high sequence similarity to short interspersed elements (SINEs). We observed similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.

genomics↗

Controlling protein nanocage assembly with hydrostatic pressure

Controlling the assembly and disassembly of nanoscale protein cages for the capture and internalisation of protein or non-proteinaceous components is fundamentally important to a diverse range of bionanotechnological applications. Here, we study the reversible, pressure-induced dissociation of a natural protein nanocage, E. coli bacterioferritin (Bfr), using synchrotron radiation small angle X-ray scattering (SAXS) and circular dichroism (CD). We demonstrate that hydrostatic pressures of 450 MPa are sufficient to completely dissociate the Bfr icositetramer into protein dimers, and the reversibility and kinetics of the reassembly process can be controlled by selecting appropriate buffer conditions. We also demonstrate that the heme B prosthetic group present at the subunit dimer interface influences the stability and pressure lability of the cage, despite its location being discrete from the inter-dimer interface that is key to cage assembly. This indicates a major cage-stabilising role for heme within this family of ferritins.

biophysics↗

Compartmentalized RNA catalysis in membrane - free coacervate protocells

Phase separation of mixtures of oppositely charged polymers provides a simple and direct route to compartmentalization via coacervation, which may have been important for driving primitive reactions as part of the RNA world hypothesis. However, to date, RNA catalysis has not been reconciled with coacervation. Here we demonstrate that RNA catalysis is viable within coacervate microdroplets and further show that these membrane-free droplets can selectively retain longer length RNAs while permitting transfer of lower molecular weight oligonucleotides.

biophysics↗