Biology subjects

Caveney, P. M.

Publications and source records attributed to Caveney, P. M..

3 recordsLinked to original sources

bioRxiv2019

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

Essential 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.

biophysics↗

bioRxiv2019

Controlling Cell-Free Gene Expression Behavior by Tuning Membrane Transport Properties

Controlled transport of molecules across boundaries for energy exchange, sensing, and communication is an essential step toward cell-like synthetic systems. This communication between the gene expression compartment and the external environment requires reaction chambers that are permeable to molecular species that influence expression. In lipid vesicle reaction chambers, species that support expression - from small ions to amino acids - may diffuse across membranes and amplify protein production. However, vesicle-to-vesicle variation in membrane permeability may lead to low total expression and high variability in this expression. We demonstrate a simple optical treatment method that greatly reduces the variability in membrane permeability. When transport across the membrane was essential for expression, this optical treatment increased mean expression level by ~6-fold and reduced expression variability by nearly two orders of magnitude. These results demonstrate membrane engineering may enable essential steps toward cell-like synthetic systems. The experimental platform described here provides a means of understanding controlled transport motifs in individual cells and groups of cells working cooperatively through cell-to-cell molecular signaling.

biophysics↗

bioRxiv2018

Synergistic interactions between confinement and macromolecular crowding spatially order transcription and translation in cell-free expression

Synergistic interactions between macromolecular crowding and confinement spatially organize transcription and translation in cells. Yet, reproducing such spatial ordering in cell-free expression platforms has proven to be elusive. Here we report crowding- and confinement-driven spatial self-organization of cell-free expression that mimics expression behavior within and around the nucleoid of prokaryotes. These experiments use Ficoll-70 to approximate cellular macromolecular crowding conditions within cell-size lipid vesicles. Intriguingly, there was an abrupt change in transcriptional dynamics when crowding reached physiologically relevant levels. Imaging experiments revealed that this change in transcriptional dynamics was coincident with localization of plasmid DNA and mRNA at the vesicle wall. Computer simulations demonstrated that crowding leads to an entropically induced attraction between plasmid DNA and the wall, causing localization of DNA near the wall at sufficiently high crowding levels. The experiments demonstrate cell-like spatial organization of translation, where translational activity is controlled by chromosomally-templated positioning of mRNA. This cell-free system provides a flexible experimental platform to probe the underlying mechanisms of self-organization of membrane-less structures in cells and the spatial control of gene expression.

synthetic biology↗