title: Large Scale Bacterial Colony Screening of Diversified FRET Biosensors.
abstract: Biosensors based on Förster Resonance Energy Transfer (FRET) between fluorescent protein mutants have started to revolutionize physiology and biochemistry. However, many types of FRET biosensors show relatively small FRET changes, making measurements with these probes challenging when used under sub-optimal experimental conditions. Thus, a major effort in the field currently lies in designing new optimization strategies for these types of sensors. Here we describe procedures for optimizing FRET changes by large scale screening of mutant biosensor libraries in bacterial colonies. We describe optimization of biosensor expression, permeabilization of bacteria, software tools for analysis, and screening conditions. The procedures reported here may help in improving FRET changes in multiple suitable classes of biosensors.
authors.affiliation: Max-Planck-Institut für Neurobiologie, Am Klopferspitz 18, Martinsried, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Litzlbauer
authors.affiliation: Max-Planck-Institut für Neurobiologie, Am Klopferspitz 18, Martinsried, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Schifferer
authors.affiliation: Max-Planck-Institut für Neurobiologie, Am Klopferspitz 18, Martinsried, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Ng
authors.affiliation: Max-Planck-Institut für Neurobiologie, Am Klopferspitz 18, Martinsried, Germany.
authors.forename: Arne
authors.initials: A
authors.lastname: Fabritius
authors.affiliation: Max-Planck-Institut für Neurobiologie, Am Klopferspitz 18, Martinsried, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Thestrup
authors.affiliation: Max-Planck-Institut für Neurobiologie, Am Klopferspitz 18, Martinsried, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Griesbeck
mesh_terms: D001419:Bacteria / Q000302:isolation & purification*; D015374:Biosensing Techniques; D031541:Fluorescence Resonance Energy Transfer; D010539:Permeability; D012984:Software