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Small regulatory RNAs (sRNAs) are short non-coding RNAs in bacteria capableof post-transcriptional regulation. sRNAs have recently gained attention astools in basic and applied sciences for example to fine-tune genetic circuitsor biotechnological processes. Even though sRNAs often have a rather simple andmodular structure, the design of functional synthetic sRNAs is not necessarilytrivial. This protocol outlines how to use computational predictions andsynthetic biology approaches to design, construct and validate synthetic sRNAfunctionality for their application in bacteria. The computational tool,SEEDling, matches the optimal seed region with the user-selected sRNA scaffoldfor repression of target mRNAs. The synthetic sRNAs are assembled using GoldenGate cloning and their functionality is subsequently validated. The protocoluses the acrA mRNA as an exemplary proof-of-concept target in Escherichia coli.Since AcrA is part of a multidrug efflux pump, acrA repression can be revealedby assessing oxacillin susceptibility in a phenotypic screen. However, in casetarget repression does not result in a screenable phenotype, an alternativevalidation of synthetic sRNA functionality based on a fluorescence reporter isdescribed.

In silico design, in vitro construction and in vivo application of synthetic small regulatory RNAs in bacteria