Establishing Continuous in Vitro DNA Replication in a Cell-Free System

In vitro implementation of stable and robust DNA replication will be key to constructing a transcription-translation based artificial cell using a bottom-up approach. Previous attempts at reconstituting isothermal DNA replication, and eventually coupling it to a cell-free TX-TL system, have used a batch mode, which impedes the self-sustainability of the system.

To achieve self-sustainability, we have developed a robust system based on rolling circle amplification using the phi29 DNA polymerase, followed by recircularization via restriction digest and ligation that sustains stable replication for multiple rounds. Implementing this replication system on a microfluidic chemostat device promises to keep the reaction at steady state by exchanging waste products for fresh reagents using tunable dilution rates. Furthermore, this approach will allow us to continuously and autonomously replicate a target genome, establishing one of the core functionalities for an artificial cell. We are hopeful that our system will not only provide a versatile platform to study long term effects on DNA replication including evolution, but will also give further insight into how the key processes of protein expression and DNA replication may be concerted in a cell and linked to the cell cycle.