Engineering of synthetic small RNAs for multi-targeting in Escherichia coli

Synthetic biology is a rapidly evolving field focused on designing and constructing novel biological systems with specific functions. A key emerging area within synthetic biology is the development of synthetic small RNAs (sRNAs). In bacteria, sRNAs typically repress translation of their target mRNAs. Typical sRNAs consist of a seed region and a scaffold region, which are modular elements that can be engineered and combined to create synthetic sRNAs. The seed region is essential for binding and repression of mRNAs, while the scaffold provides structural features, enhancing sRNA stability and facilitating interactions with proteins, such as the RNA chaperone Hfq. Synthetic sRNAs hold significant potential for various applications. To efficiently repress translation, different synthetic seed regions and scaffolds should be evaluated. Furthermore, this study aims at constructing an sRNA array for simultaneous modulation of several phenotypes in Escherichia coli. Golden Gate cloning and Modular Cloning (MoClo) were used to assemble seed regions and scaffolds to construct synthetic sRNA expression plasmids. The functionality of synthetic sRNAs was evaluated using phenotypic screens. It was found that the regulatory activity of synthetic sRNAs varies depending on the chosen seed and scaffold regions. The seed region is critical for translational repression, but we also observed that certain scaffolds are more effective for regulation of specific mRNAs. After the initial screening, sRNAs were selected to construct an sRNA array for simultaneous repression of antibiotic resistance, flagella-based motility, and biofilm formation. We predict that the presented multi-targeting approach has numerous potential applications.