Difference between revisions of "Part:BBa J63000:Experience"

Revision as of 17:50, 26 November 2007

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Applications of BBa_J63000

mCherry has been used to track protein expression and protein localization as a function of time.

User Reviews

UNIQ05fa57d83401d04c-partinfo-00000000-QINU

BBa_J63000 Caroline Ajo-Franklin

The fluorescence from mCherry can be readily visualized by microscopy when mCherry is expressed from a reasonable promoter, i.e. CTS1, MET25, in addition to very strong promoters, i.e. GAL1, ADH1. Because mCherry's absorption band only minimially overlaps with a 568 nm laser line, the signal to noise for FACS measurements is lower than microscopy, but the signal is clearly distinguishable from the noise. The main benefit of mCherry over other red fluorescent proteins is that it is monomeric and that it has a relatively short maturation time (15 min in vitro[1] and 45 min in vivo in yeast[2]). One caveat is that in addition to the full-length mCherry, several smaller and fainter MW bands appear on a western blot of a whole cell extract from Saccharomyces cerevisiae expressing the mCherry construct. When overexpressed in E. coli, the same construct give rise to only a single band on a western.
More information about the absolute expression levels, relationship between fluorescence and number of molecules, maturation rate, and photobleaching rate can be found in [2].
[1] Shaner, N.C., et al."Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein." Nat Biotechnol. 22(12):1567-72.
[2] Ajo-Franklin, C. M. and Drubin, D. A. et al. "Rational design of memory in eukaryotic cells." Genes Dev., 21(18):2271-6.

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UNIQ05fa57d83401d04c-partinfo-00000002-QINU

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 <I>Caroline Ajo-Franklin</I>
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-The fluorescence from mCherry can be readily visualized by microscopy when mCherry is expressed from a reasonable promoter, i.e. CTS1, MET25, in addition to very strong promoters, i.e. GAL1, ADH1. Because mCherry's absorption band only minimially overlaps with a 568 nm laser line, the signal to noise for FACS measurements is lower than microscopy. The main benefit of mCherry over other red fluorescent proteins is that it is monomeric and that it has a relatively short maturation time (15 min in vitro[1] and 45 min in vivo in yeast[2]). One caveat is that in addition to the full-length mCherry, several smaller and fainter MW bands appear on a western blot of a whole cell extract from Saccharomyces cerevisiae expressing the mCherry construct. When overexpressed in E. coli, the same construct give rise to only a single band on a western. <br>
+The fluorescence from mCherry can be readily visualized by microscopy when mCherry is expressed from a reasonable promoter, i.e. CTS1, MET25, in addition to very strong promoters, i.e. GAL1, ADH1. Because mCherry's absorption band only minimially overlaps with a 568 nm laser line, the signal to noise for FACS measurements is lower than microscopy, but the signal is clearly distinguishable from the noise. The main benefit of mCherry over other red fluorescent proteins is that it is monomeric and that it has a relatively short maturation time (15 min in vitro[1] and 45 min in vivo in yeast[2]). One caveat is that in addition to the full-length mCherry, several smaller and fainter MW bands appear on a western blot of a whole cell extract from Saccharomyces cerevisiae expressing the mCherry construct. When overexpressed in E. coli, the same construct give rise to only a single band on a western. <br>
 More information about the absolute expression levels, relationship between fluorescence and number of molecules, maturation rate, and photobleaching rate can be found in [2]. <br>
 [1] Shaner, N.C., et al."Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein." Nat Biotechnol. 22(12):1567-72.<br>