In a recent article published in Advanced Science, researchers have developed the START (Synthetic Trans-Acting Riboswitch with Triggering RNA) platform, a novel method for ligand-responsive gene regulation.
By combining components from bacterial riboswitches and synthetic gene regulators, the START platform provides a flexible and programmable system for biosensing, capable of detecting and responding to various ligands with high sensitivity and a broad dynamic range.
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Study: START: A versatile platform for bacterial ligand sensing with programmable performances. Image Credit: FOTOGRIN/Shutterstock.com
Background
Cells rely on their ability to recognize and respond to signaling molecules to regulate gene expression and maintain overall cellular function. This process is typically mediated by transcription factors that change shape in response to ligand binding. However, the complexity of protein interactions and difficulties in repurposing them for synthetic biology have led researchers to seek alternative methods.
Riboswitches, natural RNA elements, regulate gene expression by undergoing conformational changes when they bind specific metabolites or ligands, affecting downstream transcription or translation. On the other hand, synthetic gene regulators, like toehold switches, enable precise control of gene expression in response to engineered RNA inputs.
By combining these two approaches, the START platform creates customizable synthetic biosensors that can detect a wide variety of chemical and protein ligands.
The Current Study
The development of the START platform involved several key experimental steps. First, researchers designed and synthesized the switch and trigger RNAs, which were expressed in the Escherichia coli BL21 DE3 strain using T7 RNA polymerase. Expression was triggered by adding isopropyl β-D-1-thiogalactopyranoside (IPTG).
After growing the cells overnight in LB medium with appropriate antibiotics, the cultures were diluted and exposed to specific concentrations of target ligands like theophylline, tetracycline, or anhydrotetracycline (aTc). Once exposed to the ligands, the transcription of the switch and trigger RNAs was induced, and the cells were monitored for fluorescence and growth over time.
Flow cytometry, using a CytoFLEX LX flow cytometer, was then employed to analyze the induced cells, measuring fluorescence intensity and assessing cell populations, with around 50,000 cells analyzed per biological replicate.
Additionally, time-course measurements were conducted with a microplate reader to track cell growth and fluorescence at regular intervals. The data from these experiments were analyzed to evaluate the START platform’s performance, including its dynamic range and sensitivity to different ligands.
Results and Discussion
The results demonstrated that the START platform exhibits remarkable performance in terms of ligand detection and gene regulation. The system's dynamic range reached up to 67.29-fold, indicating a high level of sensitivity to ligand concentrations. The platform's tunable nature allows for the optimization of ligand sensitivity, making it adaptable for various applications.
Furthermore, the modularity and composability of the START design enable the seamless integration of multiple inputs, facilitating the construction of complex genetic circuits capable of performing OR, AND, and NOT logic operations.
The successful implementation of these Boolean logic gates highlights the potential of the START platform for sophisticated biosensing applications. For instance, the two-input OR gate demonstrated the ability to produce a fluorescence output in response to either of the two ligands, while the AND gate required both inputs to activate gene expression. The NOT gate showcased the capacity to inhibit gene expression in the presence of a specific ligand, further illustrating the versatility of the system.
The findings also highlight the advantages of using RNA-based regulatory elements in synthetic biology. Unlike traditional protein-based systems, RNA switches can be engineered with greater precision and flexibility, allowing for the rapid development of biosensors tailored to specific ligands.
Additionally, the START platform's reliance on RNA elements minimizes the potential for unwanted interactions that can arise from complex protein networks, thereby enhancing the reliability of the system.
Conclusion
In conclusion, the START platform represents a significant advancement in the field of synthetic biology and biosensing. By leveraging the principles of riboswitches and toehold switches, this innovative system provides a versatile and programmable approach to ligand-responsive gene regulation.
The high dynamic range and tunable sensitivity of the START platform, combined with its modularity and composability, enable the construction of complex genetic circuits capable of executing Boolean logic operations.
These features position the START platform as a powerful tool for a wide array of applications in synthetic biology, bioengineering, and beyond. As research in this area continues to evolve, the potential for the START platform to facilitate the development of sophisticated biosensors and regulatory systems will undoubtedly contribute to advancements in our understanding of cellular processes and the engineering of biological systems.
Journal Reference
Kim J., Seo M., et al. (2024). START: A versatile platform for bacterial ligand sensing with programmable performances. Advanced Science 11(1), 2402029. DOI: 10.1002/advs.202402029, https://onlinelibrary.wiley.com/doi/10.1002/advs.202402029