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US 2013/0171626 A1Published application
Methods of evaluating gene expression levels
AssigneesMassachusetts Institute of Technology, Raytheon BBN Technologies Corp.
InventorsAaron Daniel Adler, Jacob Stuart Michael Beal, Fusun Yaman-Sirin, Ron Weiss, Noah Justin Davidsohn
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Abstract
Described herein are methods of evaluating the expression levels of DNA parts encoding proteins in test circuits. In particular, the methods disclosed herein are useful to evaluate the expression of an output protein regulated by a regulatory protein-genetic element pair.
Claims20
A method of evaluating the expression level produced by a test regulatory molecule-test genetic element pair, comprising
- providing 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 co-expressed from the same genetic element responsive to the effector-regulated protein, or are expressed separately from separate copies of the genetic 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 cells at a plurality of amounts 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 the individual cells of the population of cells,
- optionally performing signal compensation on the measured levels of the input reporter protein, the output reporter protein and the constitutive reporter protein to reduce signal overlap and produce a compensated level of input reporter protein, a compensated level of output reporter protein, and a compensated level of constitutive reporter protein,
- two-dimensionally binning the cells at each amount of the input effector into a plurality of bins by the uncompensated or compensated level of constitutive reporter protein to produce a finite number of binned cells, and calculating for each bin a bin average level of constitutive reporter protein,
- optionally normalizing, for the cells in each bin, the uncompensated or compensated level of output reporter protein and/or the uncompensated or compensated level of input reporter protein by the bin average level of constitutive reporter protein to produce a normalized level of input reporter protein and/or a normalized level of output reporter protein, and
- evaluating the expression level produced by the test regulatory molecule-test genetic element pair from the uncompensated, compensated, normalized or non-normalized level of input reporter protein and output reporter protein.
10 more elements
A method of selecting three optical bandpass filter-fluorescent protein pairs for simultaneous fluorescent measurement comprising
- a) selecting a plurality of optical bandpass filters and a plurality of fluorescent proteins,
- b) measuring the emission for each of the plurality of fluorescent proteins with each of the plurality of optical bandpass filters and scaling the emission from cold to hot, wherein cold represents low emission and hot represents high emission for each optical bandpass filter-fluorescent protein combination,
- c) building a two-dimensional matrix for the scaled emission for each of the plurality of fluorescent proteins with each of the plurality of optical bandpass filters,
- d) selecting a first test fluorescent protein and identifying a first test optical bandpass filter for the first test protein that is as hot as possible for the test fluorescent protein in the two dimensional matrix while being cold for at least a second and a third test fluorescent protein to produce a first fluorescent protein-optical bandpass filter combination,
- e) repeating step d) for the second and the third test fluorescent proteins to produce second and third fluorescent protein-optical bandpass filter combinations, and
- f) selecting the first, second and third selected optical bandpass filter-fluorescent protein combinations for simultaneous measurement of three fluorescent proteins.
3 more elements
Dependencies are inferred from the claim text.