Expression of membrane-associated proteins within single emulsion cell facsimiles

Abstract

MreB is a structural membrane-associated protein which is one of the key components of the bacterial cytoskeleton. Although it plays an important role in shape maintenance of rod-like bacteria, the understanding of its mechanism of action is still not fully understood. This study shows how segmented flow and microdroplet technology can be used as a new tool for biological in vitro investigation of this protein. In this paper, we demonstrate cell-free expression in a single emulsion system to express red fluorescence protein (RFP) and MreB linked RFP (MreB-RFP). We follow the aggregation and localisation of the fusion protein MreB-RFP in this artificial cell-like environment. The expression of MreB-RFP in single emulsion droplets leads to the formation of micrometer-scale protein patches distributed at the water/oil interface.

Synthetic Plants

“…Lipid-free experiments performed with water/oil/water (w/o/w) double emulsions (Martino et al 2012a), and water/oil (w/o) droplets (Chanasakulniyom et al 2012), showed that a freshly synthesised fusion protein, comprised of MreB joined to red fluorescent protein (RFP), accumulated at the surfactant-based emulsion interface.…”

Cell‐Free Protein Synthesis in Bifunctional Hyaluronan Microgels: A Strategy for In Situ Immobilization and Purification of His‐Tagged Proteins

“…As a result, diffusion pathways as well as protein synthesis rates in vitro can strongly differ from cellular behavior in vivo . To more closely address the complex membrane‐encompassed environment of living cells in vitro, CFPS has been confined to microscopic water‐in‐oil (W/O)‐emulsions, semi‐permeable liposomes and polymersomes, coacervates, or microclays . Beyond this, hydrogel‐based materials have attracted growing attention due to their wide range of applicable natural and synthetic polymers, which can be crosslinked by bio‐orthogonal strategies .…”

Cell-free protein expression systems in microdroplets: Stabilization of interdroplet bilayers

“…1932-1058/2013/7(1)/014108/12/$30.00 V C 2013 American Institute of Physics 7, 014108-1 fluorescent proteins, a reductase enzyme, and the membrane-associated protein MreB in pl droplets-in-oil. 7,8 Once biomolecules have been enclosed in a droplet compartment, additional reagents can be supplied by merging the original droplet with a second droplet that carries the desired molecules. 9 For example, in the case of cell-free protein expression, it can be advantageous to delay the introduction of the DNA plasmid until the droplet with the ribosomal extract is at a defined position in an array, or to replenish the molecules that supply metabolic energy after a certain period of protein expression.…”