Timer fluorescent protein

pTimer is a prokaryotic expression vector that encodes DsRed1-E5, a fluorescent protein that changes color as it ages. Shortly after translation, DsRed1-E5 emits green light; its red fluorophore emerges later, hours after translation. Because of its predictable color shift, DsRed1-E5 acts as a timer, useful for monitoring changes in gene activity in living cells.

DsRed1-E5 is a mutant of the red fluorescent protein, DsRed1. The wild-type protein, DsRed, was originally referred to as drFP583. DsRed1-E5 contains two amino acid substitutions (V105A and S197T), which increase its fluorescence intensity and endow it with a distinct spectral property: As the protein ages, it changes color. When first synthesized, DsRed1-E5 is bright green (excitation & emission maxima = 483 nm & 500 nm). As time passes, the green fluorophore undergoes additional changes that cause its fluorescence to shift to longer wavelengths-when fully matured, the protein is bright red (excitation & emission maxima = 558 nm & 583 nm). In mammalian cells transfected with a Tet-inducible DsRed1-E5 expression vector, the green-to-red transition starts about 3 hours after the protein first becomes fluorescent. In addition to the amino acid replacements, DsRed1-E5's coding sequence contains a series of silent base-pair changes, which correspond to human codon-usage preferences, for high expression in mammalian cells.

pTimer is primarily intended to serve as a convenient source of the DsRed1-E5 cDNA. The DsRed1-E5 coding sequence is flanked by separate and distinct multiple cloning sites at the 5' and 3' ends so that the gene can easily be excised for use in other expression systems. Alternatively, the DsRed1-E5 coding sequence can be amplified by PCR.

The DsRed1-E5 gene was inserted in frame with the lacZ initiation codon from pUC19 so that DsRed1-E5 is expressed from the lac promoter (P_lac) in E. coli host cells. A Kozak consensus sequence is located immediately upstream of DsRed1-E5 to enhance translational efficiency should you wish to express the gene in eukaryotic systems. The entire DsRed1-E5 expression cassette in pTimer is supported by a pUC backbone, which contains a high-copy number origin of replication and an ampicillin resistance gene for propagation and selection in E. coli.

When expressed in multicellular organisms, DsRed1-E5's invariable green-to-red shift can be used as a timer to track the on-off phases of gene expression during embryogenesis and cell differentiation.

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Vector map for pTimer vectors

Vector map for pTimer vectors. As the Fluorescent Timer matures, it changes color—in a matter of hours, depending on the expression system used.

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Fluorescent timer under the control of the heat shock promoter hsp16-41 in a transgenic C. elegans embryo

Fluorescent Timer under the control of the heat shock promoter hsp16-41 in a transgenic C. elegans embryo. The embryo was heat shocked in a 33°C water bath. Promoter activity was studied during the heat shock recovery period. Green fluorescence was observed in the embryo as early as two hr into the recovery period. By 50 hr after heat shock, promoter activity had ceased, as indicated by the lack of green color.

pTimer-1 is a promoterless vector that can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the MCS located upstream of the DsRed1-E5 coding sequence. Shortly after translation, DsRed1-E5 emits green light; its red fluorophore emerges later, hours after translation. Because of its predictable color shift, DsRed1-E5 acts as a timer, useful for monitoring changes in gene activity in living cells. pTimer-1 can be used to study the on-off activity of any cis-acting regulatory element cloned into the MCS upstream of DsRed1-E5.

DsRed1-E5 is a mutant of the red fluorescent protein, DsRed1. The wild-type protein, DsRed, was originally referred to as drFP583. DsRed1-E5 contains two amino acid substitutions (V105A and S197T), which increase its fluorescence intensity and endow it with a distinct spectral property: As the protein ages, it changes color. When first synthesized, DsRed1-E5 is bright green (excitation & emission maxima = 483 nm & 500 nm). As time passes, the green fluorophore undergoes additional changes that cause its fluorescence to shift to longer wavelengths-when fully matured, the protein is bright red (excitation & emission maxima = 558 nm & 583 nm). In mammalian cells transfected with a Tet-inducible DsRed1-E5 expression vector, the green-to-red transition starts about 3 hours after the protein first becomes fluorescent. In addition to the amino acid replacements, DsRed1-E5's coding sequence contains a series of silent base-pair changes, which correspond to human codon-usage preferences, for high expression in mammalian cells.

To further increase translation efficiency in eukaryotic cells, a sequence upstream of DsRed1-E5 has been converted to a Kozak consensus sequence. SV40 polyadenylation signals downstream of the DsRed1-E5 gene direct proper processing of the 3' end of the DsRed1-E5 mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin-resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter upstream of the cassette expresses kanamycin resistance in E. coli. Without addition of a functional promoter, this vector will not express DsRed1-E5.

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Vector map for pTimer vectors

Vector map for pTimer vectors. As the Fluorescent Timer matures, it changes color—in a matter of hours, depending on the expression system used.

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Fluorescent timer under the control of the heat shock promoter hsp16-41 in a transgenic C. elegans embryo

Fluorescent Timer under the control of the heat shock promoter hsp16-41 in a transgenic C. elegans embryo. The embryo was heat shocked in a 33°C water bath. Promoter activity was studied during the heat shock recovery period. Green fluorescence was observed in the embryo as early as two hr into the recovery period. By 50 hr after heat shock, promoter activity had ceased, as indicated by the lack of green color.