Multi-Ribozyme-gRNA-Aptazyme (RGA) networks for biosensing and gene regulation

Abstract

Biological parts can be considered as molecular building blocks to assemble complex synthetic genetic networks utilizing synthetic biology. In this work, we constructed three types of multi-Ribozyme-gRNA-Aptazyme (RGA) networks for biosensing and gene regulation by linking RGA, Ribozyme-gRNA-Ribozyme (RGR) and CRISPRi systems together. We screened out two different aptazymes that fit this network and analyzed their one-layer and two-layer single-input RGAs behaviors and functions in two-inputs systems with different architectures. Overall, we built two IMPLY gates, two NIMPLY gates, one AND gate, one partial NAND gate, one conditional non-Boolean gate. The RGA shows as much as a 7.38-fold increase in ligand-dependent system-output. The interaction between RGAs that are inserted in circuits in parallel has also been reported, which contributions to the predictability of more complicated RGA networks.