Patent ID: 8809057

Claim:
A method of evaluating the expression level produced by a test regulatory molecule-test genetic element pair in vitro, comprising providing a sample having a population of cells comprising an effector transcription unit comprising a constitutive effector genetic element and a coding sequence for an effector-regulated protein, wherein the constitutive effector genetic element controls expression of the effector-regulated protein, wherein the effector-regulated protein binds a genetic element responsive to the effector-regulated protein, and wherein the binding activity of the effector-regulated protein to the genetic element responsive to the effector-regulated protein is modulated by an input effector, a test regulatory molecule transcription unit comprising the genetic element responsive to the effector-regulated protein and a coding sequence for the test regulatory molecule, wherein the genetic element responsive to the effector-regulated protein controls expression of the test regulatory molecule, an input transcription unit comprising the genetic element responsive to the effector-regulated protein and a coding sequence for an input reporter protein, wherein the genetic element responsive to the effector-regulated protein controls expression of the input reporter protein, wherein the test regulatory molecule transcription unit and the input transcription unit are on the same genetic molecule element responsive to the effector regulated protein, or are on different expressed separately from separate copies of the genetic molecules element responsive to the effector regulated protein, an output transcription unit comprising the test genetic element responsive to the test regulatory molecule and a coding sequence for an output reporter protein, wherein the test genetic element responsive to the test regulatory molecule controls expression of the output reporter protein, and a constitutive reporter protein transcription unit comprising a constitutive genetic element and a coding sequence for a constitutive reporter protein, wherein the constitutive genetic element controls expression of the constitutive reporter protein, incubating the sample of cells with an amount of the input effector for a time and under conditions sufficient to allow expression of the effector transcription unit, the test regulatory molecule transcription unit, the input reporter protein transcription unit, the output reporter protein transcription unit, and the constitutive reporter protein transcription unit, measuring the levels of the input reporter protein, the output reporter protein and the constitutive reporter protein in individual cells of the sample, two-dimensionally binning the cells to produce a finite number of binned cells, and calculating for each bin a bin average level of constitutive reporter protein, and evaluating the expression level produced by the test regulatory molecule-test genetic element pair from the level of input reporter protein and output reporter protein.