Genetically encoded division machinery for cell free synthetic biology

The de novo construction of cellular life requires, in part, the assembly of components that confer the ability to replicate. Herein we describe efforts to reconstitute parts of the Escherichia coli cell division machinery inside of water-in-oil emulsion compartments and synthetic phospholipid vesicles. The system was built with DNA and purified transcription and translation machinery housed in a compartment. A particular emphasis was placed on FtsZ, a protein that oligomerizes into a ring at the midcell and splits the cell into two. FtsZ does not contain a membrane interaction domain. In vivo, FtsZ interactions with the membrane are mediated by FtsA and ZipA. Therefore, the influence of FtsA on the behavior of FtsZ also was investigated. Fluorescently tagged constructs were used to facilitate evaluation by microscopy. The data showed that FtsZ readily assembles into rings in the presence of FtsA, thereby suggesting that the Fts system can be exploited for building a genetically encoded, self-replicating, cell-like system. We also explored additional methods of dividing compartments, such as the use of aqueous two and three phase systems.