Cell-free protein synthesis through solubilisate exchange in water/oil emulsion compartments.

Abstract

This work is aimed at finding conditions under which synthetic compartments used as cell models can fuse with each other and allow reagents contained in the different compartments to react. This goal seems to be best achieved by the use of water in oil emulsions (w/o) with dimensions in the range of 30-60 microm. In particular, cell-free EGFP (enhanced green fluorescent protein) synthesis takes place in Tween 80/Span 80 w/o emulsions, and the extent of the reaction can be monitored directly by fluorescence. The medium is mineral oil, containing 0.5 % v/v aqueous solution. Different premixing configurations of the components (plasmid, amino acids, E. Coli extract) are used and compared. The in vitro synthesis of EGFP in emulsion droplets proceeds for 1 h, and the yield is 7.5 ng microL(-1) protein. EGFP synthesis in aqueous solution takes place for at least 5 h. The yield is 10.5 ng microL(-1) protein after 1 h and 15.8 ng microL(-1) protein after 5 h. The results with the w/o emulsions show that solubilisate exchange takes place among the different water droplets, but it is not possible to demonstrate clearly that a true fusion takes place.