Biosensors Engineered from Conditionally Stable Ligand-Binding Domains

Abstract

Disclosed is a biosensor engineered to conditionally respond to the presence of specific small molecules, the biosensors including conditionally stable ligand-binding domains (LBDs) which respond to the presence of specific small molecules, wherein readout of binding is provided by reporter genes or transcription factors (TFs) fused to the LBDs.

What is claimed is: 
     
         1 . A biosensor comprising a ligand binding domain (LBD) engineered to respond conditionally to the presence of specific small molecules, the biosensor including an LBD that is fused to a reporter protein or transcription factor (TF). 
     
     
         2 . The biosensor of  claim 1 , further comprising elements suitable for use as a tool for the detection of environmental contaminants. 
     
     
         3 . The biosensor of  claim 1 , further comprising elements suitable for use as a tool for gene regulation. 
     
     
         4 . The biosensor of  claim 1 , further comprising elements suitable for use as a tool to select modifications to biosynthetic pathways that improve bioproduction yields, efficiencies or rates. 
     
     
         5 . The biosensor of  claim 1 , further comprising a scaffold including a DNA-binding domain and transcriptional activation domain. 
     
     
         6 . The biosensor of  claim 5  wherein the DNA binding domain or transcriptional activation domain is mutated to further destabilize the biosensor and increase response to the small molecule. 
     
     
         7 . The biosensor of  claim 6  wherein a known degradation sequence tag is fused to the biosensor to further destabilize the biosensor and alter the response to the small molecule. 
     
     
         8 . The biosensor of  claim 5 , wherein ligand-induced stabilization of the TF activates expression of a reporter gene. 
     
     
         9 . The biosensor of  claim 8 , wherein incorporation of an unstable ligand-binding domain into the TF scaffold generates a destabilized protein that is more readily cleared from the cell than would be a biosensor with a stable ligand binding domain. 
     
     
         10 . The biosensor of  claim 5 , wherein addition of a cognate ligand stabilizes the ligand-binding domain and increases in vivo levels of the TF, thus coupling transcriptional activation to the level of the small molecule. 
     
     
         11 . A biosensor comprising an engineered digoxigenin-binding protein, DIG10.3, fused to the DNA-binding domain of Gal4 and a VP16 activation domain. 
     
     
         12 . The biosensor of  claim 11 , which is capable of inducing reporter expression in the presence of digoxigenin. 
     
     
         13 . The biosensor of  claim 11 , further comprising DIG10.3 variants or mutants that enhance induction by several-fold. 
     
     
         14 . The biosensor of  claim 11 , wherein deletion of an ABC transporter Pdr5, enhances the sensitivity of the sensor by restricting efflux and increasing intracellular levels of exogenously added ligand. 
     
     
         15 . The biosensor of  claim 11 , wherein further mutations are introduced to alter the sensor's substrate specificity to prefer progesterone, whereby the biosensor is able to detect the biosynthesis of progesterone. 
     
     
         16 . A method of detecting small molecules for which a binding protein either exists or can be designed comprising contacting a sample suspected of containing the small molecule with a biosensor of  claim 1 . 
     
     
         17 . A method of  claim 16 , where the LBD responds conditionally to the presence of the small molecule because it has been mutated so that its stability depends on the presence of the small molecule. 
     
     
         18 . The biosensor of  claim 1 , wherein the LBD and the reporter or TF are genetically fused. 
     
     
         19 . The biosensor of  claim 1 , wherein the components are fused together post-translationally.