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An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions
Published on: March 22, 2018
Environmentally-modulated changes in fluorescence distribution in cells with oscillatory genetic network dynamics
Stephanie Portle1, Sergio Iadevaia, Ka-Yiu San
1Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX 77251-1892, United States.
Journal of Biotechnology
|May 12, 2009
Summary
Researchers explored the repressilator genetic circuit in E. coli, discovering that unexpected interactions explain its complex behaviors like bi-threshold expression and hysteresis, advancing synthetic biology understanding.
Area of Science:
- Synthetic biology
- Genetic engineering
- Microbial systems
Background:
- The repressilator is a synthetic genetic oscillator comprising three cyclically inhibiting promoter-repressor pairs.
- Previous studies indicated oscillatory behavior at the single-cell level under specific conditions.
- Understanding gene expression control in synthetic networks is crucial for robust biological circuit design.
Purpose of the Study:
- To investigate the distribution of green fluorescent protein (GFP) expression levels in E. coli cells with a repressilator network.
- To analyze extracellular control of the repressilator using anhydrotetracycline (aTc) and isopropyl-beta-d-thiogalactopyranoside (IPTG).
- To reconcile discrepancies between theoretical models and observed complex behaviors like bi-threshold effects and hysteresis.
Main Methods:
- Shake flask experiments with E. coli expressing the repressilator.
- Flow cytometry for analyzing GFP expression levels and population distributions.
- Mathematical modeling to simulate network behavior and test hypotheses.
Main Results:
- Anhydrotetracycline (aTc) induced a novel bi-threshold behavior with hysteresis, leading to three distinct steady states.
- Isopropyl-beta-d-thiogalactopyranoside (IPTG) fine-tuned expression characteristics, reducing average expression and coefficient of variation (CV).
- Experimental findings and observed multiplicity/bi-threshold behavior were not explained by the initial model, suggesting additional interactions.
Conclusions:
- Nonspecific interactions between repressors and non-cognate promoters were hypothesized to explain the observed phenomena.
- Incorporating these nonspecific interactions into the model qualitatively matched experimental results.
- Experimental validation confirmed at least four significant nonspecific interactions, revealing the genetic architecture's flexibility and the importance of these interactions in network behavior.
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