Development of fluorogen-activated and color-switchable reporter system in live bacteria

Abstract

Fluorescent labeling of proteins has been greatly important for bioimaging to study complex molecular mechanisms in cells. The yellow fluorescence-activating and absorption-shifting tag (Y-FAST) is a small size protein, which by addition of a fluorogen can be rapidly, and reversibly activated. However, an effective strategy for multiplexed imaging while allowing temporal control has been lacking. We have developed a multiplexed version of the Y-FAST protein by synthesizing different chromophores that allow fluorescence activation and spectral shifts in various wavelengths. The newly synthesized fluorogen, called gallic aldehyde-Rhodanine (GARho), can be easily produced by the simple Knoevenagel condensation reaction between rhodanine and gallolcontaining aldehyde, 3,4,5-trihydroxybenzaldehyde. This is the first gallol-derivative study in which it is engineered to be a fluorophore for spectral tuning of protein. With the previously presented scaffold protein YFAST, the GA-Rho fluorogen bound to the protein exhibits red color fluorescent emission and reversibly turns on/off by PBS washing and rebinding of related fluorogens. The effects of binding of the fluorogens to Y-FAST were characterized using the purified protein and live-bacteria expressing the protein. Furthermore, we also demonstrated emission color interchanges of Y-FAST expressed in Escherichia coli via sequential treatment of spectrally tuned red, yellow, and green color fluorogens. This system can be widely applied as an adaptive reporter with dynamic temporal changes by multi-fluorescent emissions in living bacteria. In chapter 1, introduction of fluorescent reporter system and fluorogen-activated reporter system are described. In chapter 2, I will describe that Y-FAST and gallol derivatives can combine to emit fluorescence for each wavelength, thereby showing that it can be used as a dynamic and temporally controllable reporter system with multi-fluorescent emissions in live bacteria. In conclusion, this fluorescent reported system can potentially be applied to tag various target proteins for multiplexed imaging in living cells for study of drug bindinig, functions and signal.