Developing a highly multiplexed reporter assay to study regulatory elements in bacteria

Bacteria are able to adapt to dynamic environments quickly, by regulating their gene expression in a precise manner. In addition to core regulators, such as promoters and terminators, diverse RNA-based regulatory mechanisms such as metabolite-binding riboswitches and small regulatory RNAs (sRNAs) help bacteria fine-tune their expression profiles. These RNA-based regulatory elements are often located in the 5’ untranslated regions (5’ UTR) of many genes of environmental and clinical bacteria. Thanks to the DNA sequencing revolution the sequence of hundreds-of-thousands of putative regulatory elements is available. Yet, to understand their roles in biology requires that we go beyond mapping and into functional analysis. Yet, the throughput of approaches for mechanistic characterization never scaled up to the efficiency of feature mapping, and currently each regulatory element is studied one by one. The aim of my project is to create a generic system to allow studying many thousands of such regulatory elements at the same time. This approach is based on massively parallel reporter assays coupled with next generation sequencing. If successful, this method will allow us to experimentally study the activities of thousands of currently uncharacterized regulatory elements and shed light on the regulatory capacity of clinically and biotechnologically important bacteria.