Dynamics of sequestration-based gene regulatory cascades

Area of Science:

• Synthetic Biology
• Systems Biology
• Gene Regulation

Background:

• Gene regulatory cascades are fundamental to biological processes but challenging to analyze in native systems.
• Synthetic biology offers a powerful approach to dissecting regulatory cascade properties through engineered circuits.
• Sequestration-based regulatory cascades, a common motif, remain less explored, particularly regarding component control over output dynamics.

Purpose of the Study:

• To construct and quantitatively analyze the longest known sequestration-based regulatory cascade.
• To elucidate how cascade components collectively influence output dynamics in a synthetic system.
• To investigate the impact of negative feedback on the response times of sequestration-based cascades.

Main Methods:

• Construction of a four-protein (ExsADCE) sequestration-based cascade in Escherichia coli, derived from Pseudomonas aeruginosa regulators.
• Computational modeling to predict the influence of unbounded transcriptional activator (ExsA) concentration on output dynamics.
• Experimental validation by systematically varying cascade length and regulator synthesis rates, including introducing negative feedback.

Main Results:

• Computational analysis revealed complex control of output dynamics by free ExsA concentration.
• Experimental results confirmed ExsC's role in rapid responses via ExsD sequestration, and ExsD's role in increasing response times.
• Introduction of indirect negative feedback significantly reduced the cascade's response time.

Conclusions:

• The study presents the longest sequestration-based cascade, offering quantitative insights into this regulatory mechanism.
• Findings demonstrate how specific components (ExsC, ExsD) modulate response speed and dynamics.
• The results provide crucial understanding of sequestration-based cascades with negative feedback, a prevalent motif in natural biological systems.