Steering prokaryotic gene expression using engineered riboswitches

Several techniques originating from synthetic biology have a big impact on the field of metabolic engineering. These methods are successfully being used to reprogram cell behaviour for example by constructing genetic circuits or introducing heterologous biosynthetic pathways. In this context, specific and controlled gene expression based on the presence of a certain molecule is of great value. Such regulatory elements are abundantly present in nature. Most of the engineered devices use the post-transcriptional level (RNA) to exert control over gene expression because of the versatility and designability of RNA. Despite these advantages, the engineering process of these RNA devices remains very challenging and relies mostly on high-throughput screening. The current lack of in-depth knowledge limits the widespread application of these promising devices. Here, we propose a framework for the design and construction of riboswitches. These RNA devices exhibit translational control by conditionally blocking the ribosome binding site (RBS) when the specific molecule is absent and releasing this blockage when the same molecule becomes available. For this purpose, riboswitches are composed of two structurally linked domains: an aptamer and a gene expression platform. Upon the ligand binding the aptamer part structurally rearranges which leads to RNA conformational changes in the expression platform. This unblocks the RBS which allows translation of the gene located downstream. In this work, several types of riboswitches are designed and evaluated using a fluorescent protein as reporter gene. Since gene regulation by riboswitches requires conditional base-pairing, RNA secondary structure prediction algorithms are used during the design phase. As such, computational tools are used to determine suitable RNA sequences with the desired properties. After designing different riboswitch configurations the various designs were tested in vivo to determine their performance. From the performance of the different designs systemic rules are derived which can lead to a generic guideline for the design of riboswitches in general. Ultimately, this approach can be used for the design of tailor made riboswitches with excellent performance. The authors would like to acknowledge Inbiose for their contributions