Implementation And Simulation Of Phosphorylation-Based Insulator In Transcription-Translation Platform

The operational amplifier (OPAMP) is a very useful insulation module in electric circuits to avoid loading effect (retroactivity). In synthetic biological circuits, we also have the same retroactivity problem, in which the biomolecular systems are not always modular due to downstream components. The output of the upstream component will be affected as the downstream component sequesters that output, which in turn impedes the process of constructing more complex biocircuits. To address this obstacle, the retroactivity needs to be attenuated by implementing a similar OPAMP device using biocircuits. Previous theoretical papers suggested a potential function of a phosphorylation based circuit in providing the feature of attenuating retroactivity. Here we presented a successful prototyping and implementation of such a phosphorylation-based insulator (PBI) in an in vitro cell-free transcription-translation system (TXTL). We demonstrated that retroactivity also exists in TX-TL system, if not stronger, by testing a simple negative regulation circuit. Besides we showed that the TX-TL system contains all the protein, DNA components and other resources required for the PBI circuit to work properly. We then demonstrated that the PBI circuit helps minimizing the loading effect to less than 10% compared to control circuit. With this preliminary PBI circuit design, attenuation of retroactivity while connecting two modules in vitro becomes possible. In concert with another paper from our group (E. Yeung, S. Guo, R. Murray QBIO2014) which used system identification to estimate all three essential parameters in a simplified PBI model, we showed that the simulations …