"Fluorescent Timer": Protein That Changes Color with Time-Reference-Cited by-同舟云学术

"Fluorescent Timer": Protein That Changes Color with Time

Published:2000-11-24 Issue:5496 Volume:290 Page:1585-1588
ISSN:0036-8075
Container-title:Science
language:en
Short-container-title:Science

Author:

Terskikh Alexey¹,Fradkov Arkady²,Ermakova Galina²,Zaraisky Andrey²,Tan Patrick¹,Kajava Andrey V.³,Zhao Xiaoning⁴,Lukyanov Sergey²,Matz Mikhail²,Kim Stuart¹,Weissman Irving¹,Siebert Paul⁴

Affiliation:

1. School of Medicine, Stanford University, Stanford, CA 94305, USA.

2. Institute of Bioorganic Chemistry, Russian Academy of Science, 117871 Moscow, Russia.

3. Center for Molecular Modeling, Center for Information Technology, NIH, Building 12A, Bethesda, MD 20892, USA.

4. Clontech Laboratories, 1020 East Meadow Circle, Palo Alto, CA 94303, USA.

Abstract

We generated a mutant of the red fluorescent protein drFP583. The mutant (E5) changes its fluorescence from green to red over time. The rate of color conversion is independent of protein concentration and therefore can be used to trace time-dependent expression. We used in vivo labeling with E5 to measure expression from the heat shock–dependent promoter in Caenorhabditis elegans and from the Otx-2 promoter in developing Xenopus embryos. Thus, E5 is a “fluorescent timer” that can be used to monitor both activation and down-regulation of target promoters on the whole-organism scale.

Publisher

American Association for the Advancement of Science (AAAS)

Subject

Multidisciplinary

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4. Random mutagenesis was performed with Diversity PCR Random Mutagenesis kit (Clontech) according to the manufacturer's protocol optimized for three to four mutations per 1000 base pairs. PCR products were cloned into pQE-30/Bam HI/Hind III vector. Escherichia coli DH5-α (Clontech) were transformed by electroporation in 10% glycerol with ligation mixture and were grown on Luria broth (LB)/agar/Amp plates with 0.1 mM isopropyl-β- d -thiogalactopyranoside at 37°C overnight. Colonies (up to 2000 to 5000 per plate) were screened visually using a fluorescent microscope (Karl Zeiss) with 31001 filter set (Chroma). For study of fluorescence kinetics bacteria harboring the recombinant plasmid were grown overnight on the LB plates at high density scraped resuspended in ice-cold phosphate-buffered saline (PBS) and sonicated. The lysate was cleared by centrifugation at 4°C and the protein was purified from the supernatant on ice with TALON resin (Clontech). All spectra were measured on purified proteins with a LS50B Luminescence Spectrometer (Perkin-Elmer).

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