Prototyping and Implementation of a Novel Feedforward Loop in a Cell-Free Transcription-Translation System and Cells

Building novel synthetic biological devices is a time-consuming task because of the lengthy cellbased testing and optimization processes. Recent progress made in the cell-free field suggests that the utilization of mathematical models and cell-free transcription-translation testing platforms to systematically design and test novel synthetic biocircuits may help streamline some of the processes. Here we present a study of building a novel functional biological network motif from scratch with the aid of the mathematical modeling and the cell-free prototyping. In this work, we demonstrated that we were able to make a 3-promoter feedforward circuit from a concept to a working biocircuit in cells within a month. We started with performing simulations with a cell-free transcriptiontranslation simulation toolbox. After verifying the feasibility of the circuit design, we used a fast assembling method to build the constructs and used the linear DNAs directly in the cell-free system for prototyping. After additional tests and assemblies, we implemented the circuit in plasmid forms in cells and showed that the in vivo results were consistent with the simulations and the outcomes in the cell-free platform. This study showed the usefulness of modeling and prototyping in building synthetic biocircuits and that we can use these tools to help streamline the process of circuit optimizations in future studies. . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/123190 doi: bioRxiv preprint first posted online Apr. 2, 2017;