Part:BBa_M50009:Design
mRuby3 fluorescent protein
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 16
Design Notes
FRET is a sensitive phenomenon and must be optimized with ideal fluorescent proteins to obtain the best results. For FRET to occur, it is important that the donor fluorescent protein has an emission spectra that overlaps significantly with the absorption spectra of the acceptor fluorescent protein. However, you must also ensure that the acceptor and donor emissions are sufficiently separated since overlaps in emission can. This can be quantified by calculating the FRET efficiency of the fluorescent pair (https://www.amherst.edu/media/view/106141/original/FRET.pdf). In our device, we implemented FRET using a mRuby3-cometGFP pair. This pairing is not as ideal as other GFPs, like mClover3 which was developed in a previous study by Bajar et al. However, we were constrained by a limit on the number of base pairs allowed in our construct, since our glucose-binding protein was over 1200 bp. Having to include mRuby3 in our unique ordered construct, we chose to use a standard cometGFP that would not delay the manufacturing time of our construct.
Source
mRuby3 was developed by Bryce T. Bajar et al. in their recent publication of “Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting” (http://www.nature.com/articles/srep20889). It is a variant of the previously developed mRuby2, being blue-shifted, more photostable, brighter, and more optimized for mammalian cells.