Orthogonal riboswitches as tools for controlling gene expression in bacteria

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Gene expression is the process by which DNA sequence information is first transcribed in to messenger RNA (mRNA), and then translated to produce proteins. The expression of genes is tightly controlled (regulated) by a number of mechanisms, which affect the timing and levels by which the gene products (RNAs and proteins) are synthesised in response to environmental conditions and other signals. For example, it was recently discovered that certain metabolites in cells can turn gene expression on or off by triggering switches present within mRNA. These so called riboswitches are found in all domains of life, and are particularly wide spread in bacteria. Typically these switches regulate the production, degradation or transport of specific metabolites. For example the add A-riboswitch binds adenine to activate translation of the mRNA that encodes the enzyme adenine deaminase, which degrades adenine. Through this feedback mechanism the bacteria can control the cellular levels of adenine a key building block in DNA synthesis. Recently we succeeded in re-engineering (or rewiring) add A-riboswitches, so that they are no longer triggered by the natural metabolites present in the cell, but instead can be controlled by the addition of various synthetic molecules (ligands). In this project we aim to develop new orthogonal riboswitches through further genetic manipulation and by developing new and more effective synthetic ligands which will allow more precise and dynamic control of gene expression in bacteria. In addition to riboswitches that can activate gene expression on binding synthetic ligands, we will also apply our strategy to re-engineer riboswitches which can block gene expression in response to selected synthetic molecules. We will also couple multiple switches together, in a tandem arrangement, to enable a more digital control of gene expression. For example we propose to develop a genetic switch which can be turned on with one ligand, activating gene expression, before being switched off with a second distinct ligand. In addition we will also show how the mutually orthogonal riboswitches can be employed to affect the simultaneous and differential control of multiple genes in bacteria. The new genetic switches we develop could be used to study the function of genes in bacteria. In the case of pathogenic bacteria, which cause disease, expression tools based on orthogonal riboswitches could be used to validate new targets for antibiotics that could lead to the development of new antimicrobial treatments. The riboswitch expression tools could also be used to aid production of proteins in bacteria, which could include therapeutically important proteins (biopharmaceuticals) or industrial important enzymes (biocatalysts). In addition, orthogonal riboswitches could be used to control multiple genes which encode metabolic pathways leading to natural product based drugs, biofuels and other commercially important products which are emerging as targets for synthetic biology. During this project we will be working to develop orthogonal riboswitches with the required properties that will allow us to demonstrate their utility in these important applications.