Imperial College London
Alternate

DrMarkFriddin

Faculty of Engineering, Dyson School of Design Engineering

Imperial College Research Fellow
 
 
 
//

Contact

 

m.friddin

 
 
//

Location

 

Dyson BuildingSouth Kensington Campus

//

Summary

 

Publications

Citation

BibTex format

@article{Friddin:2013:10.1063/1.4791651,
author = {Friddin, MS and Morgan, H and de, Planque MR},
doi = {10.1063/1.4791651},
journal = {Biomicrofluidics},
title = {Cell-free protein expression systems in microdroplets: Stabilization of interdroplet bilayers},
url = {http://dx.doi.org/10.1063/1.4791651},
volume = {7},
year = {2013}
}
Download

RIS format (EndNote, RefMan)

TY  - JOUR
AB  - Cell-free protein expression with bacterial lysates has been demonstrated to produce soluble proteins in microdroplets. However, droplet assays with expressed membrane proteins require the presence of a lipid bilayer. A bilayer can be formed in between lipid-coated aqueous droplets by bringing these into contact by electrokinetic manipulation in a continuous oil phase, but it is not known whether such interdroplet bilayers are compatible with high concentrations of biomolecules. In this study, we have characterized the lifetime and the structural integrity of interdroplet bilayers by measuring the bilayer current in the presence of three different commercial cell-free expression mixtures and their individual components. Samples of pure proteins and of a polymer were included for comparison. It is shown that complete expression mixtures reduce the bilayer lifetime to several minutes or less, and that this is mainly due to the lysate fraction itself. The fraction that contains the molecules for metabolic energy generation does not reduce the bilayer lifetime but does give rise to current steps that are indicative of lipid packing defects. Gel electrophoresis confirmed that proteins are only present at significant amounts in the lysate fractions and, when supplied separately, in the T7 enzyme mixture. Interestingly, it was also found that pure-protein and pure-polymer solutions perturb the interdroplet bilayer at higher concentrations; 10% (w/v) polyethylene glycol 8000 (PEG 8000) and 3 mM lysozyme induce large bilayer currents without a reduction in bilayer lifetime, whereas 3 mM albumin causes rapid bilayer failure. It can, therefore, be concluded that the high protein content of the lysates and the presence of PEG polymer, a typical lysate supplement, compromise the structural integrity of interdroplet bilayers. However, we established that the addition of lipid vesicles to the cell-free expression mixture stabilizes the interdroplet bilayer, allowing
AU  - Friddin,MS
AU  - Morgan,H
AU  - de,Planque MR
DO  - 10.1063/1.4791651
PY  - 2013///
SN  - 1932-1058
TI  - Cell-free protein expression systems in microdroplets: Stabilization of interdroplet bilayers
T2  - Biomicrofluidics
UR  - http://dx.doi.org/10.1063/1.4791651
UR  - http://hdl.handle.net/10044/1/40144
VL  - 7
ER  -
Download