Optogenetic Synchronization and Entrainment of a Synthetic Genetic Oscillator

Główne pojęcia

Integrating an optogenetic module into a repressilator circuit enables the use of light to synchronize, entrain, and detune oscillations in gene expression within single cells or entire populations.

Streszczenie

The authors introduce the "optorepressilator", a synthetic genetic oscillator that combines a repressilator circuit with an optogenetic module. This allows them to control the oscillations using light as an external stimulus.
Key highlights:

The optorepressilator consists of a repressilator circuit with an additional light-inducible copy of the LacI repressor. This enables light to act as a "zeitgeber" to synchronize and entrain the oscillations.

Experiments show that a population of optorepressilators can be synchronized by transient green light exposure, resetting the phases of individual oscillators.

Periodic light pulses can entrain the optorepressilators to oscillate indefinitely at the frequency of the external signal, even when it differs from the natural frequency of the oscillator.

Detuning experiments reveal multiple synchronization regimes, with the system exhibiting different locking behaviors depending on the frequency and amplitude of the external light signal.

The authors develop a simple mathematical model that quantitatively captures the phase-sensitive response of the optorepressilator to light pulses, explaining the observed synchronization and entrainment phenomena.

The optorepressilator demonstrates how integrating optogenetic control into synthetic genetic circuits can enable precise spatiotemporal programming of gene expression dynamics, with potential applications in areas like biological timekeeping and coordination of cellular behaviors.

Statystyki

"Estimates for the LacI dissociation constant K vary between 0.01 0.1 nM [33]."
"RBS substitution leads to a 27-fold decrease in reporter production (Fig. 3b), while relocation of the light-driven cassette to the genome reduces expression by 57-fold."
"The combination of weak RBS and genome insertion results in a gene expression range below our instrument sensitivity (estimated combined fold reduction of 1500)."

Cytaty

"Light, for example, can penetrate bioreactors and orchestrate gene expression in a population of uncoupled oscillators."
"Integrating experiments and mathematical modeling, we show that the entrainment mechanism is robust and can be understood quantitatively from single cell to population level."

Kluczowe wnioski z

Light-driven synchronization of optogenetic clocks

by Cannarsa,M. ... o www.biorxiv.org 10-24-2023

https://www.biorxiv.org/content/10.1101/2023.10.24.563722v2

Głębsze pytania

How could the optorepressilator system be extended to enable more complex spatiotemporal control of gene expression, such as generating traveling waves or oscillatory patterns

To extend the optorepressilator system for more complex spatiotemporal control of gene expression, several modifications and additions can be considered. One approach could involve incorporating spatial gradients of light intensity to create traveling waves of gene expression. By varying the intensity of light across different regions, cells could respond differently based on their position, leading to a wave-like pattern of gene expression propagation. This could be achieved by implementing a setup with spatially controlled light sources or using microfluidic devices to create gradients of light exposure. Additionally, introducing feedback mechanisms that adjust light intensity based on the gene expression levels in neighboring cells could further enhance the generation of traveling waves.
Another strategy to achieve oscillatory patterns could involve coupling multiple optorepressilator systems in a network. By interconnecting individual optorepressilators through light-sensitive interactions, it would be possible to synchronize their oscillations and create more complex dynamics. This network of optogenetic clocks could exhibit emergent behaviors such as synchronized oscillations, phase shifts, and even chaotic patterns, depending on the connectivity and parameters of the system. By carefully designing the interactions between the optorepressilators and the light inputs, researchers could generate intricate spatiotemporal patterns of gene expression across a population of cells.

What are the potential limitations or challenges in translating this optogenetic synchronization approach to more complex natural or synthetic biological systems

Translating the optogenetic synchronization approach to more complex biological systems or synthetic circuits may face several limitations and challenges. One potential limitation is the scalability of the system to larger populations of cells or more intricate genetic networks. As the complexity of the system increases, the design and optimization of light-sensitive components, such as the optogenetic modules and promoters, become more challenging. Ensuring precise control over gene expression in a diverse and dynamic biological environment could also pose difficulties, as factors like cell-to-cell variability, environmental conditions, and genetic noise may impact the robustness of synchronization.
Another challenge lies in the integration of the optogenetic system with endogenous biological processes or existing synthetic circuits. Coordinating the optorepressilator with other genetic elements or regulatory networks within the cell requires careful consideration of crosstalk, interference, and compatibility. Balancing the light inputs with the natural regulatory mechanisms of the cell to achieve desired gene expression patterns without disrupting essential cellular functions is crucial for successful implementation.
Furthermore, the optimization of parameters such as light intensity, duration, and timing for different biological contexts or applications may require extensive experimental validation and fine-tuning. Variability in light penetration, cell response to light, and system dynamics could affect the reproducibility and reliability of the synchronization approach in diverse settings. Addressing these challenges will be essential for the broader application of optogenetic synchronization in complex biological systems.

Could the principles underlying the optorepressilator be applied to develop light-controlled oscillators in other domains, such as chemical or physical systems, to study synchronization phenomena more broadly

The principles underlying the optorepressilator system can indeed be applied to develop light-controlled oscillators in various domains beyond biological systems. In the realm of chemical systems, optically controlled reactions or catalytic processes could be designed using similar concepts of light-inducible promoters, repressors, and feedback loops. By incorporating photoresponsive molecules or materials that undergo chemical transformations upon light exposure, researchers can create oscillatory behavior or temporal control over reaction kinetics.
In physical systems, light-controlled oscillators could be engineered using optomechanical devices, where light inputs modulate mechanical vibrations or oscillations. By integrating light-sensitive actuators or sensors into mechanical systems, researchers can achieve precise control over frequency, phase, and amplitude of oscillatory patterns. This approach could find applications in fields such as photonics, acoustics, and signal processing, enabling the development of light-driven oscillators for communication, sensing, and information processing.
Overall, the optorepressilator concept demonstrates the versatility and potential of optogenetic tools for controlling dynamic systems with light inputs. By adapting these principles to different domains, researchers can explore synchronization phenomena, pattern formation, and dynamic control in a wide range of disciplines beyond biology.