scholarly journals Molecular Transport across Lipid Membranes Controls Cell-Free Expression Level and Dynamics

2019 ◽  
Author(s):  
Patrick M. Caveney ◽  
Rosemary M. Dabbs ◽  
William T. McClintic ◽  
C. Patrick Collier ◽  
Michael L. Simpson
Keyword(s):  
Gene Expression ◽  
Membrane Transport ◽  
Molecular Species ◽  
Lipid Membranes ◽  
Molecular Transport ◽  
Lipid Vesicles ◽  
Cellular Systems ◽  
Control Of Gene Expression ◽  
External Resources ◽  
Synthetic Cell

SummaryEssential steps toward synthetic cell-like systems require controlled transport of molecular species across the boundary between encapsulated expression and the external environment. When molecular species (e.g. small ions, amino acids) required for expression (i.e. expression resources) may cross this boundary, this transport process plays an important role in gene expression dynamics and expression variability. Here we show how the location (encapsulated or external) of the expression resources controls the level and the dynamics of cell-free protein expression confined in permeable lipid vesicles. Regardless of the concentration of encapsulated resources, external resources were essential for protein production. Compared to resource poor external environments, plentiful external resources increased expression by ~7-fold, and rescued expression when internal resources were lacking. Intriguingly, the location of resources and the membrane transport properties dictated expression dynamics in a manner well predicted by a simple transport-expression model. These results suggest membrane engineering as a means for spatio-temporal control of gene expression in cell-free synthetic biology applications and demonstrate a flexible experimental platform to understand the interplay between membrane transport and expression in cellular systems.

scholarly journals CTCF and cellular heterogeneity

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Gang Ren ◽  
Keji Zhao
Keyword(s):  
Gene Expression ◽  
Fundamental Property ◽  
Cellular Systems ◽  
Cellular Heterogeneity ◽  
Control Of Gene Expression ◽  
Homogeneous Population ◽  
Cellular Processes ◽  
Underlying Mechanisms ◽  
Cell Variation ◽  
Promoter Interaction

Abstract Cellular heterogeneity, which was initially defined for tumor cells, is a fundamental property of all cellular systems, ranging from genetic diversity to cell-to-cell variation driven by stochastic molecular interactions involved all cellular processes. Different cells display substantial variation in gene expression and in response to environmental signaling even in an apparently homogeneous population of cells. Recent studies started to reveal the underlying mechanisms for cellular heterogeneity, particularly related to the states of chromatin. Accumulating evidence suggests that CTCF, an important factor regulating chromatin organization, plays a key role in the control of gene expression variation by stabilizing enhancer–promoter interaction.

scholarly journals Shaping liposomes by cell-free expressed bacterial microtubules

2021 ◽  
Author(s):  
Christophe Danelon ◽  
Marileen Dogterom ◽  
Johannes Kattan ◽  
Anne Doerr
Keyword(s):  
Lipid Membranes ◽  
Cell Biology ◽  
De Novo ◽  
Lipid Vesicles ◽  
Translation System ◽  
Dna Templates ◽  
Cytoskeletal Network ◽  
Synthetic Cell ◽  
Directional Transport ◽  
Shape Changes

Genetic control over a cytoskeletal network inside lipid vesicles offers a potential route to controlled shape changes and DNA segregation in synthetic cell biology. Bacterial microtubules (bMTs) are protein filaments found in bacteria of the genus Prosthecobacter. They are formed by the tubulins BtubA and BtubB which polymerize in the presence of GTP. Here, we show that the tubulins BtubA/B can be functionally expressed from DNA templates in a reconstituted transcription-translation system, thus providing a cytosol-like environment to study their biochemical and biophysical properties. We found that bMTs spontaneously interact with lipid membranes and display treadmilling. When compartmentalized inside liposomes, de novo synthesized BtubA/B tubulins self-organize into cytoskeletal structures of different morphologies. Moreover, bMTs can exert a pushing force on the membrane and deform liposomes, a phenomenon that can be reversed by light-activated disassembly of the filaments. Our work establishes bMTs as a new building block in synthetic biology. In the context of creating a synthetic cell, bMTs could help shape the lipid compartment, establish polarity or directional transport, and assist the division machinery.

scholarly journals Posttranscriptional Control of Gene Expression in Yeast

1998 ◽  
Vol 62 (4) ◽  
pp. 1492-1553 ◽  
Author(s):  
John E. G. McCarthy
Keyword(s):  
Gene Expression ◽  
Mrna Decay ◽  
Molecular Mechanisms ◽  
Genomic Sequence ◽  
Current Knowledge ◽  
Cellular Systems ◽  
Posttranscriptional Control ◽  
Control Of Gene Expression ◽  
Translational Initiation ◽  
Wide Range

SUMMARY Studies of the budding yeast Saccharomyces cerevisiae have greatly advanced our understanding of the posttranscriptional steps of eukaryotic gene expression. Given the wide range of experimental tools applicable to S. cerevisiae and the recent determination of its complete genomic sequence, many of the key challenges of the posttranscriptional control field can be tackled particularly effectively by using this organism. This article reviews the current knowledge of the cellular components and mechanisms related to translation and mRNA decay, with the emphasis on the molecular basis for rate control and gene regulation. Recent progress in characterizing translation factors and their protein-protein and RNA-protein interactions has been rapid. Against the background of a growing body of structural information, the review discusses the thermodynamic and kinetic principles that govern the translation process. As in prokaryotic systems, translational initiation is a key point of control. Modulation of the activities of translational initiation factors imposes global regulation in the cell, while structural features of particular 5′ untranslated regions, such as upstream open reading frames and effector binding sites, allow for gene-specific regulation. Recent data have revealed many new details of the molecular mechanisms involved while providing insight into the functional overlaps and molecular networking that are apparently a key feature of evolving cellular systems. An overall picture of the mechanisms governing mRNA decay has only very recently begun to develop. The latest work has revealed new information about the mRNA decay pathways, the components of the mRNA degradation machinery, and the way in which these might relate to the translation apparatus. Overall, major challenges still to be addressed include the task of relating principles of posttranscriptional control to cellular compartmentalization and polysome structure and the role of molecular channelling in these highly complex expression systems.

scholarly journals A tunable dual-input system for on-demand dynamic gene expression regulation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Elisa Pedone ◽  
Lorena Postiglione ◽  
Francesco Aulicino ◽  
Dan L. Rocca ◽  
Sandra Montes-Olivas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Stability ◽  
Mammalian Cells ◽  
Dynamic Range ◽  
Embryonic Stem ◽  
Cellular Systems ◽  
Control Of Gene Expression ◽  
Cell Functions ◽  
Input System ◽  
Gene Expression Control

Abstract Cellular systems have evolved numerous mechanisms to adapt to environmental stimuli, underpinned by dynamic patterns of gene expression. In addition to gene transcription regulation, modulation of protein levels, dynamics and localization are essential checkpoints governing cell functions. The introduction of inducible promoters has allowed gene expression control using orthogonal molecules, facilitating its rapid and reversible manipulation to study gene function. However, differing protein stabilities hinder the generation of protein temporal profiles seen in vivo. Here, we improve the Tet-On system integrating conditional destabilising elements at the post-translational level and permitting simultaneous control of gene expression and protein stability. We show, in mammalian cells, that adding protein stability control allows faster response times, fully tunable and enhanced dynamic range, and improved in silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to modulate levels of signalling pathway components in mouse Embryonic Stem Cells.

scholarly journals A tunable dual-input system for ‘on-demand’ dynamic gene expression regulation

2018 ◽  
Author(s):  
Elisa Pedone ◽  
Dan L. Rocca ◽  
Lorena Postiglione ◽  
Francesco Aulicino ◽  
Sandra Montes-Olivas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Protein Stability ◽  
Small Molecules ◽  
Cellular Systems ◽  
Signalling Pathway ◽  
Control Of Gene Expression ◽  
Protein Levels ◽  
Cell Functions ◽  
Input System

AbstractCellular systems have evolved numerous mechanisms to finely control signalling pathway activation and properly respond to changing environmental stimuli. This is underpinned by dynamic spatiotemporal patterns of gene expression. Indeed, in addition to gene transcription and translation regulation, modulation of protein levels, dynamics and localization are also essential checkpoints that govern cell functions. The introduction of tetracycline-inducible promoters has allowed gene expression control using orthogonal small molecules, facilitating rapid and reversible manipulation to study gene function in biological systems. However, differing protein stabilities means this solely transcriptional regulation is insufficient to allow precise ON-OFF dynamics, thus hindering generation of temporal profiles of protein levels seen in vivo. We developed an improved Tet-On based system augmented with conditional destabilising elements at the post-translational level that permits simultaneous control of gene expression and protein stability. Integrating these properties to control expression of a fluorescent protein in mouse Embryonic Stem Cells (mESCs), we found that adding protein stability control allows faster response times to changes in small molecules, fully tunable and enhanced dynamic range, and vastly improved microfluidic-based in-silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to finely modulate levels of signalling pathway components in stem cells.

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