In vitro synthetic biology in continuous modeView all posters
In vitro transcription and translation reactions can be programmed with DNA templates to produce networks of genetic regulators. In the classical batch format, however, network complexity and reaction time are limited because synthesis rates constantly decrease while precursors are consumed and products accumulate. To be able to produce genetically encoded oscillations during an in vitro transcription and translation reaction, we had to keep synthesis rates at a high level for extended periods of time and to remove reaction products. To achieve these goals we performed reactions in a continuous mode. Fresh reagents were added in short, regular time intervals into nanoliter-scale reactors on a microfluidic chip, where they displaced part of the old reaction volume. Simultaneously, mRNA and protein concentrations could be followed over time by fluorescence measurements. In our device mRNA and protein synthesis proceeded for over 24 hours at steady state. The system was highly predictable and controllable. We showed that it is possible to express a wide range of genetic regulators in our device including a RNA polymerase, transcription factors, RNA based regulators and a protease. In a continuous reaction mode, these genetic regulators can be combined into dynamic regulatory networks. This we demonstrated by constructing a transcription and translation based in vitro oscillator. Our device allowed us to tightly control synthesis and dilution rates to produce oscillations, which only occurred in a small range of the possible parameter space. Dynamic and more complex in vitro networks can be constructed using continuous reaction conditions. This approach will be useful for testing DNA constructs before they are introduced into cells. Furthermore, it raises the question whether there are any limits in the complexity of systems that can be implemented in vitro, and may ultimately allow the assembly of the cytoplasmic functions of an artificial cell.