Enhancing fluorescent protein photostability through robot-assisted photobleaching

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Engineering

Background:

• Directed evolution of fluorescent proteins necessitates efficient screening methods for large genetic variant libraries.
• Photostability is a critical parameter for the utility of fluorescent proteins in various biological applications.

Purpose of the Study:

• To develop a cost-effective system for screening fluorescent protein variants for improved photostability.
• To engineer enhanced variants of the yellow fluorescent protein mCitrine.

Main Methods:

• Construction of a photobleaching robot using a Lego Mindstorms Ev3 set and a xenon arc lamp.
• Development of a patterned illumination technique for even irradiance across Petri dishes.
• Screening of fluorescent protein libraries for photostability improvements.

Main Results:

• Successful development of a robust and low-cost screening system for fluorescent protein photostability.
• Identification of Citrine2, a variant of mCitrine with significantly improved photostability.
• Citrine2 retains high fluorescent brightness comparable to the original mCitrine.

Conclusions:

• The described photobleaching robot system is effective for screening fluorescent protein variants.
• The engineered Citrine2 protein offers enhanced photostability for biotechnological applications.
• This method provides a valuable tool for advancing fluorescent protein engineering.