Bacterial Biosensors Based on Ligand-Dependent Stability | AIChE

Bacterial Biosensors Based on Ligand-Dependent Stability

Authors 

Brandsen, B. - Presenter, University of Washington
Mattheisen, J., University of Washington
Fields, S., University of Washington
Biosensors enable cells to sense and respond to intracellular metabolites or proteins. They can be engineered to be sensitive and selective, making them useful for orthogonal control of gene expression, environmental monitoring, and metabolic engineering. Many bacterial biosensors are based on allosteric transcription factors that bind ligand and increase expression of a reporter gene. Engineering allosteric transcription factors to selectively bind to new ligands is challenging, however, and frequently results in broken allostery. We developed a new Escherichia coli biosensor that functions based on ligand-dependent stabilization. In this design, a ligand-binding domain is fused to a DNA-binding domain and a transcription-activating domain, and the stability of the biosensor is engineered to be ligand-dependent. In the absence of ligand, rapid degradation of the destabilized ligand-binding domain limits expression of a reporter gene, but in the presence of ligand, stabilization of the biosensor increases reporter gene expression. Using error-prone PCR mutagenesis and selection, we obtained a DNA-binding defective variant of LacI, the lac repressor, that functions as a biosensor when fused to the Zif268 DNA-binding domain and the omega subunit of RNA Polymerase. The biosensor detects the lactose mimic IPTG and activates expression of a Zif268-dependent reporter gene, allowing growth in selective media. The biosensor enables ~5-fold increased growth rate after induction with 1 mM IPTG. The ligand-binding domain of the biosensor contains six amino acid mutations, one of which is primarily responsible for biosensor behavior. We are currently developing biosensors that bind to polyketide natural products, and we intend to use these biosensors in conjunction with high-throughput mutagenesis to enable selection of novel polyketides.