Efficient cell-free translation from diverse human cell types DOI Creative Commons
Jana Ziegelmüller,

Nikolaos Kouvelas,

Priyanka Thambythurai

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 16, 2024

Motivation The difficulties in producing cell-free translation systems from different cell types limit the ability to study regulatory mechanisms that depend on biological contexts. Developing tailored diverse would be instrumental investigating cell-type-specific translational control, co- and post-translational modifications, viral manipulation strategies. Our method addresses this gap by providing a scalable adaptable solution for high-quality lysates reflect specific needs of types. Summary Cell-free are indispensable studying protein synthesis, enabling researchers explore regulation across Here, we present preparing translation-competent range frequently used human lines using dual centrifugation. We optimized lysis conditions adherent suspension cells, HEK-293 (adherent suspension), HeLa, SH-SY5Y, U2OS cells. results demonstrate cell-specific factors influence efficiency, with HeLa cells showing highest activity. also observed sensitivity varies between lines, underscoring importance fine-tuning parameters efficient production. provides robust approach generating lysates, broadening application vitro mechanisms.

Language: Английский

Peptide-Mediated Liquid-Liquid Phase Separation and Biomolecular Condensates DOI

Guangle Li,

Chengqian Yuan, Xuehai Yan

et al.

Soft Matter, Journal Year: 2025, Volume and Issue: 21(10), P. 1781 - 1812

Published: Jan. 1, 2025

Peptide-mediated liquid–liquid phase separation (LLPS) underpins the formation of dynamic biomolecular condensates, regulated by diverse molecular interactions, and highlights potential applications in drug delivery synthetic biology.

Language: Английский

Citations

2
Dynamic RNA synthetic biology: new principles, practices and potential DOI Creative Commons
Yueyi Li, Aníbal Arce, Tyler Lucci

et al.

RNA Biology, Journal Year: 2023, Volume and Issue: 20(1), P. 817 - 829

Published: Dec. 3, 2023

An increased appreciation of the role RNA dynamics in governing function is ushering a new wave dynamic synthetic biology. Here, we review recent advances engineering systems across molecular, circuit and cellular scales for important societal-scale applications environmental human health, bioproduction. For each scale, introduce core concepts folding at that then discuss technologies incorporating these concepts, covering approaches to riboswitches, ribozymes, origami, strand displacement circuits, biomaterials, biomolecular condensates, extracellular vesicles cells. Considering nature within design process promises spark next innovation will expand scope impact biotechnologies.

Language: Английский

Citations

11
Chemical reactions regulated by phase-separated condensates DOI Creative Commons
Sudarshana Laha, Jonathan Bauermann, Frank Jülicher

et al.

Physical Review Research, Journal Year: 2024, Volume and Issue: 6(4)

Published: Nov. 4, 2024

Phase-separated liquid condensates can spatially organize and thereby regulate chemical processes. However, the physicochemical mechanisms underlying such regulation remain elusive as intramolecular interactions responsible for phase separation give rise to a coupling between diffusion reactions at nondilute conditions. Here, we derive theoretical framework that decouples of scaffold molecules from reaction kinetics diluted clients. As result, volume client partitioning coefficients become control parameters, which enables us dissect impact phase-separated on reactions. We apply this two processes show how affect yield reversible initial rate simple assembly process. In both cases, find an optimal condensate respective property is maximal. Our work be applied experimentally quantify condensed phases alter in systems biology unravel biomolecular biochemistry living cells. Published by American Physical Society 2024

Language: Английский

Citations

4
Constructing mechanosensitive signalling pathways de novo in synthetic cells DOI Creative Commons
James W. Hindley

Biochemical Society Transactions, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 21, 2025

Biological mechanotransduction enables cells to sense and respond mechanical forces in their local environment through changes cell structure gene expression, resulting downstream function. However, the complexity of living systems obfuscates mechanisms mechanotransduction, hence study these processes vitro has been critical characterising function existing mechanosensitive membrane proteins. Synthetic are biomolecular compartments that aim mimic organisation, functionality behaviours biological systems, represent next step development models. In recent years, channels have incorporated into synthetic create de novo signalling pathways. Here, I will discuss developments, from molecular parts used construct pathways, such potential future directions engineering mechanotransduction. The recapitulation biology facilitate an improved understanding interactions across length scales, whilst simultaneously generating new biotechnologies can be applied as diagnostics, microreactors therapeutics.

Language: Английский

Citations

0
Programming biological communication between distinct membraneless compartments DOI
Boyang Ji,

H. Pan,

Zhi‐Gang Qian

et al.

Nature Chemical Biology, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 5, 2025

Language: Английский

Citations

0
Converging frontiers in biomolecular condensate and synthetic cell research DOI Creative Commons
Madelief A. M. Verwiel, Nadia A. Erkamp, Jan C. M. van Hest

et al.

npj Biomedical Innovations., Journal Year: 2025, Volume and Issue: 2(1)

Published: May 3, 2025

Language: Английский

Citations

0
Hybrid Protocells based on Coacervate-Templated Fatty Acid Vesicles combine Improved Membrane Stability with Functional Interior Protocytoplasm DOI Open Access
Jessica Lee, Fatma Pir Cakmak, Richard Booth

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Aug. 8, 2024

Abstract Prebiotically-plausible compartmentalization mechanisms include membrane vesicles formed by amphiphile self-assembly and coacervate droplets liquid-liquid phase separation. Both types of structures form spontaneously can be related to cellular motifs in today’s living cells. As prebiotic compartments, they have complementary capabilities, with coacervates offering excellent solute accumulation membranes providing superior boundaries. Herein, we describe protocell models constructed spontaneous encapsulation mixed fatty acid/phospholipid purely acid membranes. Coacervate-supported over a range lipid compositions, properties impacted charge-charge interactions between Vesicles coacervate-templated assembly exhibited profoundly different permeability than traditional or blended without interiors, particularly the presence Mg 2+ ions. While were disrupted addition 25 mM MgCl 2 , corresponding coacervate-supported remained intact impermeable externally-added solutes even . With more robust membrane, fluorescein diacetate (FDA) hydrolysis, which is commonly used for cell viability assays, could performed inside model due simple diffusion FDA then following coacervate-mediated abiotic hydrolysis fluorescein.

Language: Английский

Citations

2
Membraneless Compartmentalization of Cell‐Free Transcription‐Translation by Polymer‐Assisted Liquid–Liquid Phase Separation DOI Creative Commons
Ziane Izri, Vincent Noireaux

Small, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 6, 2024

Abstract Living cells use liquid–liquid phase separation (LLPS) to compartmentalize metabolic functions into mesoscopic‐sized droplets. Deciphering the mechanisms at play in LLPS is therefore critical understanding structuration and of subcellular level. Although observed achieved a significant degree control vivo, reconstitution integrating advanced biological functions, such as gene expression, has been so far limited vitro. cell‐free transcription‐translation (TXTL) reactions require multi‐step experimental approaches that lack biomimetic have relatively poor efficacy, thus limiting their usage engineered systems synthetic cells. Here polymer‐assisted TXTL are reported single‐pot one‐step compartmentalization model complex system obtain without using solvents or surfactants. occurs by adding biocompatible polymers poly(ethylene glycol), poly(vinyl alcohol), dextran reaction, remains highly active. These serve partitioning agents localize droplets rich dextran. Cytoplasmic membrane‐interacting proteins synthesized preferentially inside these droplets, either uniformly interface, depending on nature. The LLPS‐TXTL presented this work step toward design membraneless active organelles.

Language: Английский

Citations

1
Hybrid Protocells Based on Coacervate‐Templated Fatty Acid Vesicles Combine Improved Membrane Stability with Functional Interior Protocytoplasm DOI Creative Commons
Jessica Lee, Fatma Pir Cakmak, Richard Booth

et al.

Small, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 14, 2024

Abstract Prebiotically‐plausible compartmentalization mechanisms include membrane vesicles formed by amphiphile self‐assembly and coacervate droplets liquid–liquid phase separation. Both types of structures form spontaneously can be related to cellular motifs in today's living cells. As prebiotic compartments, they have complementary capabilities, with coacervates offering excellent solute accumulation membranes providing superior boundaries. Herein, protocell models constructed spontaneous encapsulation mixed fatty acid/phospholipid purely acid are described. Coacervate‐supported over a range lipid compositions, properties impacted charge–charge interactions between membranes. Vesicles coacervate‐templated assembly exhibit profoundly different permeability than traditional or blended without interior, particularly the presence magnesium ions (Mg 2+ ). While disrupted addition Mg , corresponding coacervate‐supported remain intact impermeable externally‐added solutes. With more robust membrane, fluorescein diacetate (FDA) hydrolysis, which is commonly used for cell viability assays, performed inside model due simple diffusion FDA then following coacervate‐mediated abiotic hydrolysis fluorescein.

Language: Английский

Citations

1
Efficient cell-free translation from diverse human cell types DOI Creative Commons
Jana Ziegelmüller,

Nikolaos Kouvelas,

Priyanka Thambythurai

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 16, 2024

Motivation The difficulties in producing cell-free translation systems from different cell types limit the ability to study regulatory mechanisms that depend on biological contexts. Developing tailored diverse would be instrumental investigating cell-type-specific translational control, co- and post-translational modifications, viral manipulation strategies. Our method addresses this gap by providing a scalable adaptable solution for high-quality lysates reflect specific needs of types. Summary Cell-free are indispensable studying protein synthesis, enabling researchers explore regulation across Here, we present preparing translation-competent range frequently used human lines using dual centrifugation. We optimized lysis conditions adherent suspension cells, HEK-293 (adherent suspension), HeLa, SH-SY5Y, U2OS cells. results demonstrate cell-specific factors influence efficiency, with HeLa cells showing highest activity. also observed sensitivity varies between lines, underscoring importance fine-tuning parameters efficient production. provides robust approach generating lysates, broadening application vitro mechanisms.

Language: Английский

Citations

0