For simultaneous mutagenesis at several positions of the mTagBFP gene the overlap-extension approach has been applied [19] (link). After mutagenesis a mixture of the mutants was electroporated into LMG194 host cells (Invitrogen)
Protein expression in the library was induced overnight at 37°C with 0.002% arabinose. Next morning, expressing bacteria were washed with PBS and then diluted with PBS for FACS sorting to optical density of 0.02 at 600 nm. MoFlo cell sorter (Dako) equipped with standard argon, krypton and argon-krypton mixed-gas lasers was used. To select the most photostable clones the library was illuminated before FACS using LED array at 405 nm for 15 min. About 10 sizes of the library were sorted on the FACS with 407 nm of krypton excitation line and 450/65 nm emission filter. The collected bright blue bacterial cells were rescued in rich SOC medium at 37°C for one hour, and then plated on Petri dishes with 0.02% arabinose. The next day, the dishes were analyzed with Leica MZ16F fluorescence stereomicroscope using a custom blue filter set (390/40 nm exciter, 460/40 nm emitter) from Chroma. To select the most photostable colonies, dishes were illuminated with 405 nm LEDs (80 mW/cm2) for 15 min, and images were acquired before and after illumination. For further analysis, 20 to 50 brightest and photostable clones were selected and analyzed using Olympus IX81 inverted microscope equipped with a 200 W metal-halide lamp (Prior), 100×1.4 NA oil objective lens (UPlanSApo, Olympus), and 390/40 nm excitation and 460/40 nm emission filters. At this stage photobleaching of the blue bacterial clones was checked. The best clones were applied for sequencing.
Protein expression in the library was induced overnight at 37°C with 0.002% arabinose. Next morning, expressing bacteria were washed with PBS and then diluted with PBS for FACS sorting to optical density of 0.02 at 600 nm. MoFlo cell sorter (Dako) equipped with standard argon, krypton and argon-krypton mixed-gas lasers was used. To select the most photostable clones the library was illuminated before FACS using LED array at 405 nm for 15 min. About 10 sizes of the library were sorted on the FACS with 407 nm of krypton excitation line and 450/65 nm emission filter. The collected bright blue bacterial cells were rescued in rich SOC medium at 37°C for one hour, and then plated on Petri dishes with 0.02% arabinose. The next day, the dishes were analyzed with Leica MZ16F fluorescence stereomicroscope using a custom blue filter set (390/40 nm exciter, 460/40 nm emitter) from Chroma. To select the most photostable colonies, dishes were illuminated with 405 nm LEDs (80 mW/cm2) for 15 min, and images were acquired before and after illumination. For further analysis, 20 to 50 brightest and photostable clones were selected and analyzed using Olympus IX81 inverted microscope equipped with a 200 W metal-halide lamp (Prior), 100×1.4 NA oil objective lens (UPlanSApo, Olympus), and 390/40 nm excitation and 460/40 nm emission filters. At this stage photobleaching of the blue bacterial clones was checked. The best clones were applied for sequencing.
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