Light-directed evolution of dynamic, multi-state, and computational protein functionalities DOI Open Access
Vojislav Gligorovski, Marco Labagnara, Sahand Jamal Rahi

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Март 2, 2024

Abstract Directed evolution is a powerful method in biological engineering. Current approaches draw on time-invariant selection mechanisms, ideal for evolving steady-state properties such as enzymatic activity or fluorescence intensity. A fundamental problem remains how to continuously evolve dynamic, multi-state, computational functionalities, e.g., on-off kinetics, state-specific activity, stimulus-responsiveness, switching and logic capabilities. These require pressure all of the states protein interest (POI) transitions between them. We realized that optogenetics cell cycle oscillations could be leveraged novel directed paradigm (‘optovolution’) germane this need: designed signaling cascade budding yeast where optogenetic input switches POI off (0) (1) states. In turn, controls Cdk1 cyclin, which re-engineered system essential one stage but poisonous another. Thus, cyclin must oscillate (1-0-1-0…) proliferation. system, can act efficiently dynamics, transient states, input-output relations every cycle. Further, controlling pacemaker, light, directs tunes pressures. Optovolution vivo, continuous, self-selecting, efficient. first evolved two systems, relay 0/1 output: obtained 25 new variants widely used LOV transcription factor El222 were stronger, less leaky, green-and red-responsive. The latter was conjectured impossible domains needed multiplexing lowering phototoxicity. Evolving PhyB-Pif3 we discovered loss YOR1 makes supplementing expensive unstable chromophore phycocyanobilin (PCB) unnecessary. Finally, demonstrate generality by destabilized rtTA factor, performs an AND operation transcriptional doxycycline input. coveted, difficult-to-change functionalities evolvable.

Язык: Английский

Light-directed evolution of dynamic, multi-state, and computational protein functionalities DOI Open Access
Vojislav Gligorovski, Marco Labagnara, Sahand Jamal Rahi

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Март 2, 2024

Abstract Directed evolution is a powerful method in biological engineering. Current approaches draw on time-invariant selection mechanisms, ideal for evolving steady-state properties such as enzymatic activity or fluorescence intensity. A fundamental problem remains how to continuously evolve dynamic, multi-state, computational functionalities, e.g., on-off kinetics, state-specific activity, stimulus-responsiveness, switching and logic capabilities. These require pressure all of the states protein interest (POI) transitions between them. We realized that optogenetics cell cycle oscillations could be leveraged novel directed paradigm (‘optovolution’) germane this need: designed signaling cascade budding yeast where optogenetic input switches POI off (0) (1) states. In turn, controls Cdk1 cyclin, which re-engineered system essential one stage but poisonous another. Thus, cyclin must oscillate (1-0-1-0…) proliferation. system, can act efficiently dynamics, transient states, input-output relations every cycle. Further, controlling pacemaker, light, directs tunes pressures. Optovolution vivo, continuous, self-selecting, efficient. first evolved two systems, relay 0/1 output: obtained 25 new variants widely used LOV transcription factor El222 were stronger, less leaky, green-and red-responsive. The latter was conjectured impossible domains needed multiplexing lowering phototoxicity. Evolving PhyB-Pif3 we discovered loss YOR1 makes supplementing expensive unstable chromophore phycocyanobilin (PCB) unnecessary. Finally, demonstrate generality by destabilized rtTA factor, performs an AND operation transcriptional doxycycline input. coveted, difficult-to-change functionalities evolvable.

Язык: Английский

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