iGEM 2012
Why make logic circuits with strand displacement? How does strand displacement work? Strand displacement reactions work in vivo!
Sensing mRNA Levels using Strand Displacement Strand Displacement NOT Gate Design Making short RNAs in vivo to use in circuits

Project Description

The limited availability of promoters, genes, and repressors, along with the difficulty in assembling and delivering large DNA plasmids bottleneck advances in sophistication of genetic circuits in mammalian systems. In contrast, sophistication of in vitro synthetic DNA circuits has grown exponentially through the mechanism of toehold-mediated strand displacement. These circuits demonstrate digital logic with reliable, modular, and scalable behaviors and maintain a small base-pair footprint.

The raw processing power of these strand displacement circuits has been trapped in the test tube, sequestered from the traditional protein-based sensing, processing, and actuation method of synthetic biology. With the adaptation of strand displacement-based information processing, the application space of synthetic biology circuits will become larger and more accessible.

Our project leverages strand displacement to create a processing technology that supports multi-input sensing, sophisticated information processing, and precisely-regulated actuation in mammalian cells. We designed and tested a novel fully-functioning DNA NOT gate, which enables complete logic operation. In addition, we used RNA strand displacement to sense cellular mRNA. We also demonstrated our ability to produce short RNAs in vivo.

Most importantly, we demonstrated that toehold-mediated strand displacement in RNA can occur in mammalian cells. This, combined with our feasibility studies outlined in the above paragraph, shows that strand displacement is a viable information-processing technology. We envision in vivo RNA strand displacement as a new foundation for scaling up complexity in engineered biological systems, with applications in biosynthesis, biomedical diagnostics and therapeutics.