Development and Characterization of Improved Red Fluorescent Protein Variants

Manna, Premashis

Graduate Thesis Or Dissertation

Development and Characterization of Improved Red Fluorescent Protein Variants Öffentlichkeit Deposited

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Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/s7526c55z

Abstract

Aequorea victoria-based green fluorescent proteins and their blue, cyan and red counterparts offer unprecedented advantage as biological markers owing to their genetic encodability and straightforward expression in different organisms. Fluorescent proteins are characterized with complex photo-kinetics due to the presence of light-induced non-fluorescent or dark states which are responsible for their fluorescence intermittency or 'blinking'. We developed time- and frequency-domain techniques for probing the kinetics involving dark state conversion (DSC) and ground state recovery (GSR) in red fluorescent proteins (RFPs). Ensemble-level DSC and GSR kinetics in FPs were presented in the context of their single molecule fluorescence behaviors. Although significant advancements have been made towards improving the key photo-physical properties of the red fluorescent proteins (RFPs), they continue to perform sub-optimally compared to their green or cyan counterparts. We developed a high-throughput microfluidic screening and sorting platform to generate improved RFP variants by engineering their excited state lifetimes. Using this microfluidic system, mCherry and FusionRed mutants were developed with higher in-vivo brightness and fusion efficiency. We employed a novel rational design for the enhancement of brightness in RFPs through engineering of their radiative rates and extinction coefficients. This enabled us to produce bright mutants those out-performed the existing red fluorescent proteins.

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