Implementation and Characterization of Dynamic Genetic Networks in Vitro

Transcription and translation (TX-TL) can be performed in vitro, outside of cells, allowing the assembly and analysis of genetic networks. This approach to engineering biological networks in a less complex and more controllable environment could one day allow rapid prototyping of network designs before implementing them in living cells. Furthermore, the in vitro approach provides insight into how natural biological systems are built and is instructive to define the rules for engineering biological systems from bottom up. Despite progress in engineering TX-TL mixes for higher yields and longer synthesis times it remains challenging to implement complex genetic networks, such as oscillators, in vitro. The reason is that the reactions are usually performed in a batch format, where reaction products accumulate and synthesis rates decline over time. We addressed the problems associated with batch reactions by developing a microfluidic chip with nanoliter-scale reactors that exchange reagents at dilution rates matching those of dividing bacteria. In these nano-reactors we can run TX-TL reactions in continuous mode keeping synthesis rates at constant steady state levels for more than 30h. The setup allows close control over the reaction conditions such as dilution rates and DNA template concentration, and to monitor mRNA and reporter protein levels in real time. We can test any genetic program of our choice just by adding the DNA templates coding for the desired functions. We implemented diverse regulatory mechanisms on the transcriptional, translational, and posttranslational levels, including RNA polymerases, transcriptional repression, translational activation, and proteolysis. As a proof of concept for this reactor-based approach to engineering genetic networks we designed and implemented a novel genetic oscillator. Its network architecture consists of a positive feedback loop coupled to delayed negative feedback. Varying dilution rates and DNA template concentrations we mapped its phase diagram showing that steady state conditions were necessary to produce oscillations. The period of oscillations could be tuned by dilution rate. To demonstrate that in vitro synthetic biology is useful for prototyping of dynamic genetic networks, we compared the behavior of biomolecular ring oscillators in a cell-free framework and Escherichia coli. We implemented and characterized the “repressilator”, a three-node negative feedback oscillator in vitro. We then used our cell-free framework to engineer novel three-node, four-node, and five-node negative feedback architectures going from the characterization of circuit components to the rapid analysis of complete networks. We validated our cell-free approach by transferring the three-node and five-node oscillators to E. coli, resulting in robust and synchronized oscillations reflecting our in vitro observations. Our results demonstrate that comprehensive circuit characterization can be rapidly performed in a cell-free system and that these in vitro results have direct applicability in vivo. The reactor-based approach to in vitro synthetic biology, which was developed in this thesis, will thus enable a quantitative and more complete characterization of synthetic and natural genetic networks.