17 research outputs found
Protein Sizing with 15 nm Conical Biological Nanopore YaxAB
Nanopores are promising single-molecule tools for the electrical identification and sequencing of biomolecules. However, the characterization of proteins, especially in real-time and in complex biological samples, is complicated by the sheer variety of sizes and shapes in the proteome. Here, we introduce a large biological nanopore, YaxAB for folded protein analysis. The 15 nm cis-opening and a 3.5 nm trans-constriction describe a conical shape that allows the characterization of a wide range of proteins. Molecular dynamics showed proteins are captured by the electroosmotic flow, and the overall resistance is largely dominated by the narrow trans constriction region of the nanopore. Conveniently, proteins in the 35-125 kDa range remain trapped within the conical lumen of the nanopore for a time that can be tuned by the external bias. Contrary to cylindrical nanopores, in YaxAB, the current blockade decreases with the size of the trapped protein, as smaller proteins penetrate deeper into the constriction region than larger proteins do. These characteristics are especially useful for characterizing large proteins, as shown for pentameric C-reactive protein (125 kDa), a widely used health indicator, which showed a signal that could be identified in the background of other serum proteins. </p
Monodisperse Uni- and Multicompartment Liposomes
Liposomes
are self-assembled phospholipid vesicles with great potential
in fields ranging from targeted drug delivery to artificial cells.
The formation of liposomes using microfluidic techniques has seen
considerable progress, but the liposomes formation process itself
has not been studied in great detail. As a result, high throughput,
high-yielding routes to monodisperse liposomes with multiple compartments
have not been demonstrated. Here, we report on a surfactant-assisted
microfluidic route to uniform, single bilayer liposomes, ranging from
25 to 190 ÎĽm, and with or without multiple inner compartments.
The key of our method is the precise control over the developing interfacial
energies of complex W/O/W emulsion systems during liposome formation,
which is achieved via an additional surfactant in the outer water
phase. The liposomes consist of single bilayers, as demonstrated by
nanopore formation experiments and confocal fluorescence microscopy,
and they can act as compartments for cell-free gene expression. The
microfluidic technique can be expanded to create liposomes with a
multitude of coupled compartments, opening routes to networks of multistep
microreactors
Microfluidic Assembly of Monodisperse Vesosomes as Artificial Cell Models
Vesosomes are nested
liposomal structures with high potential as
advanced drug delivery vehicles, bioreactors and artificial cells.
However, to date no method has been reported to prepare monodisperse
vesosomes of controlled size. Here we report on a multistep microfluidic
strategy for hierarchically assembling uniform vesosomes from dewetting
of double emulsion templates. The control afforded by our method is
illustrated by the formation of concentric, pericentric and multicompartment
liposomes. The microfluidic route to vesosomes offers an exceptional
platform to build artificial cells as exemplified by the in vitro
transcription in “nucleus” liposomes and the mimicry
of the architecture of eukaryotic cells. Finally, we show the transport
of small molecules across the nucleic envelope via insertion of nanopores
into the bilayers
Microfluidic Assembly of Monodisperse Vesosomes as Artificial Cell Models
Vesosomes are nested
liposomal structures with high potential as
advanced drug delivery vehicles, bioreactors and artificial cells.
However, to date no method has been reported to prepare monodisperse
vesosomes of controlled size. Here we report on a multistep microfluidic
strategy for hierarchically assembling uniform vesosomes from dewetting
of double emulsion templates. The control afforded by our method is
illustrated by the formation of concentric, pericentric and multicompartment
liposomes. The microfluidic route to vesosomes offers an exceptional
platform to build artificial cells as exemplified by the in vitro
transcription in “nucleus” liposomes and the mimicry
of the architecture of eukaryotic cells. Finally, we show the transport
of small molecules across the nucleic envelope via insertion of nanopores
into the bilayers
Sigma factor-mediated tuning of bacterial cell-free synthetic genetic oscillators
Cell-free transcription-translation provides a simplified prototyping environment to rapidly design and study synthetic networks. Despite the presence of a well characterized toolbox of genetic elements, examples of genetic networks that exhibit complex temporal behavior are scarce. Here, we present a genetic oscillator implemented in an E. coli-based cell-free system under steady-state conditions using microfluidic flow reactors. The oscillator has an activator-repressor motif that utilizes the native transcriptional machinery of E. coli: the RNAP and its associated sigma factors. We optimized a kinetic model with experimental data using an evolutionary algorithm to quantify the key regulatory model parameters. The functional modulation of the RNAP was investigated by coupling two oscillators driven by competing sigma factors, allowing the modification of network properties by means of passive transcriptional regulation
Macromolecularly Crowded Protocells from Reversibly Shrinking Monodisperse Liposomes
The compartmentalization
of cell-free gene expression systems in
liposomes provides an attractive route to the formation of protocells,
but these models do not capture the physical (crowded) environment
found in living systems. Here, we present a microfluidics-based route
to produce monodisperse liposomes that can shrink almost 3 orders
of magnitude without compromising their stability. We demonstrate
that our strategy is compatible with cell-free gene expression and
show increased protein production rates in crowded liposome protocells
Macromolecularly Crowded Protocells from Reversibly Shrinking Monodisperse Liposomes
The compartmentalization
of cell-free gene expression systems in
liposomes provides an attractive route to the formation of protocells,
but these models do not capture the physical (crowded) environment
found in living systems. Here, we present a microfluidics-based route
to produce monodisperse liposomes that can shrink almost 3 orders
of magnitude without compromising their stability. We demonstrate
that our strategy is compatible with cell-free gene expression and
show increased protein production rates in crowded liposome protocells