Development of a Set of Standardized Toehold Switch Riboregulators and Fluorescent Reporter Proteins for Genetic Circuit Prototyping.

Abstract

The goal of this research was the creation and characterization of a new library of Type IIS standardized biological parts that can be used to construct expression devices for reporter proteins with precise and reliable control. Toehold switches are one such element of control. As de-novo designed translational riboregulators, they can regulate ribosomal activity on an mRNA, thereby affecting post-transcriptional gene expression. Toehold switches act as a cognate pair of RNAs: the switch RNA interferes with ribosomal activity until a target RNA complementarily binds to the switch to allow for ribosomal binding and gene expression. For this research, eleven toehold switches were designed and constructed to be triggered by the target mRNA of a specific fluorescent reporter protein. The toehold switches were assembled with fluorescent reporter proteins, and these constructs were experimentally validated in a plate reader to determine toehold switch leakiness: demonstrating their efficacy in preventing translation of the downstream fluorescent protein. In total, 26 new basic parts, over 90 transcriptional units, eight multi-transcriptional units, and nine new assembly vectors were created that adhere to iGEM’s Type IIS assembly standard, based on the previously defined MoClo and Loop standards. These parts and vectors allow for quick and reliable one-pot assembly of entire transcriptional units, or assemblies of multiple transcriptional units. This new library of Type IIS characterized part samples allows for quick prototyping of genetic circuit designs.