Reporter Systems
From FreeBio
Some strategies for speeding up our fluorescent reporting:
- Bimolecular Fluorescence Complementation (BiFC) link - two components of a fluorophore are fused to separate partners; when the partners interact, the fluorophore comes together and fluoresces. This way reporters could be expressed constitutively and be activated by a faster means than transcription; how to hook this up to our genetic circuits is another matter. Chris 15:45, 7 Jun 2005 (EDT)
- mCherry, a more efficient version of RFP. From Shaner et al., Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotech 2004. pdf This is a monomeric fluorophore which matures from nascent polypeptide to folded and ready to shine in 15 minutes rather than several hours. This will dramatically cut down on the turnaround between activating transcription of the reporter and visual results, and best of all it appears a lovely shade of purple in visible light. There's a whole series of these improved fluorophores in different shades (e.g., mHoneydew and mTangerine) but mCherry matures the fastest. The BioWire team should definitely consider this one since the output has to race against signal diffusion, and the SketchaDoodle team can certainly appreciate all the pretty colors. Several of these variants are already in the Registry - however these are for use in yeast, and we will need to get our hands on bacterial plasmid versions. (Thanks to Ira for the tip!) Chris 15:45, 7 Jun 2005 (EDT)
- The molecule mCherry was derived from, mRFP1, reaches 100% fluorescence in ~2h after translation in E. coli, according to the paper in which it was described. mCherry was measured to mature 4 times as fast, meaning that once expressed we should be able to see the color within 30 minutes. The rate of expression will depend on the promoter, the RBS, and the size of the gene. link --Chris 15:33, 10 Jun 2005 (EDT)
- A temperature-sensitive bacterial luciferase. Folding is inhibited at 37 °C, normal at 23 °C. Could be useful for the thermal coaster, but may not be right for the BactaDoodle. See Escher et al. in PNAS 1989, Bacterial luciferase alpha beta fusion protein is fully active as a monomer and highly sensitive in vivo to elevated temperature. pdf (More quantitative data on temperature-sensitive luciferase variants here pdf Chris 23:55, 8 Jun 2005 (EDT)
