Abstract

Single Molecule Microfluidic Methods to Characterise Vesicle-based Cell Mimetics

Ali Salehi-Reyhani, PhD Student, Imperial College London

Bottom-up synthetic biology attempts to derive a fundamental understanding of how to build a functional genome from constituent biochemistry. Cell mimics are built or synthesised by bringing together different types of biochemical machinery, from proteins through to lipids and DNA, in user defined combinations. Methods to produce artificial cells that incorporate novel functionality are becoming ever more capable; however, methods to measure their molecular constituents post-manufacture are lacking. This must be addressed if cell mimics are to serve as quantitative models of biology.[1] We present an ultra-sensitive single molecule microfluidic method to precisely and quantitatively measure the concentration of biomolecules (DNA, RNA, protein) in single artificial cells. Giant unilamellar vesicles produced by phase transfer of an inverted emulsion to encapsulate protein were assessed. The method allowed us to precisely determine the encapsulation efficiency per vesicle, which we discovered to vary significantly between 0.01% and 10% with a weak dependence on vesicle size. The results suggest what may be deduced as heterogeneity in an artificial cell’s behaviour may be in fact due, at least in part, to technical artefacts in their production.