Engineering CRISPR/dCas based toggle switches in Escherichia coli

Synthetic biology is a field that leverages genetic engineering methods to create modular components and novel biological interactions inside microorganisms. An example of this is the creation of bistable genetic circuits called toggle switches inside E. coli bacteria which can effectively store 1 bit of information in a living organism. While current implementations of genetic toggle switches are typically based on a LacI and TetR repressor pairs, these promoter-repressor pairs suffer from low orthogonality and limited programmability. Recent advances in synthetic biology have highlighted the advantages of using of catalytically 'dead' version of Cas proteins that can selectively bind to specific DNA sequences to create logic ON/OFF switches (Specht et al. 2020, Rouches et al. 2022). In this work, we first develop a thermodynamic model to investigate parameters that affects the bistability of toggle switches based on CRISPR binding. We later use a massively parallel assay called X-seq to efficiently characterize these parameters for hundreds of toggle-switch constructs. Additionally, to visualize the kinetic features of our CRISPR-based toggle switch constructs, we also took advantage of single-cell imaging technique and a microfulidic chip platform to demonstrate the actuation dynamics of a toggle switch in real-time. Our results also uncover different classes of activity that result from modulating the growth rate of the bacteria by supplying minimal and rich media.