Building a DNA segregation system for a synthetic cell

Beatriz Orozco Monroy  and  Reza Amini Hounejani

In this project, our team aims at building a DNA segregation system based on bacterial cytoskeletal filaments recruiting a cell-free gene expression mechanism. In this regard, we test different existing options including bacterial microtubules, TubZRC segregation system, ParMRC partitioning system, and PhuZ fialments. A third possible option for each of these systems is to combine any of the two systems to build a hybrid segregation machine which is controllable by light.

Figure 1 Artificial DNA segregation model. The proteins are expressed with a PURE system in a liposome and elongate the liposome upon polymerization in the presence of GTP. The expressed adapter proteins bind to either a filament or a replicated DNA. The illumination of the sample with blue light triggers the association of the two adapter proteins and hence the binding of the DNA to the filaments. Growing (Shrinking) filaments push (pull) the bound DNA towards cell poles and the DNA is released after the illumination is stopped.

One way of dealing with the overwhelming complexity of cells, is to use reductionist approaches to gain a sufficient level of understanding of cellular phenomena. This can be achieved via two routes: top-down and bottom-up, each of which has its own advantages and disadvantages. Top-down approaches rely on removing the components of a system one by one and study the resulting effect on the system, whereas bottom-up approaches consist of the reconstitution of a specific cellular system or process using minimal components. While reconstitutions of complex systems are challenging and success has been limited, they provide an excellent tool to dissect the role of individual components under highly controlled conditions. One of the long-investigated mysteries of biology is the exact mechanism of cell division and DNA segregation and associated regulation pathways. So far minimal systems have been reconstituted that mimic prokaryotic DNA segregation but to date there has been no reconstitution of DNA segregation mediated by a bipolar eukaryotic mitotic spindle.