Engineering RNA Aptamer-Based Gene Circuits for Control of Microbial Interactions

The ability to precisely and accurately control genetic circuits has far reaching implications for the engineering of robust and safe microbial systems with desired outputs. To achieve that goal, we propose to develop a scalable and generalizable pipeline for the development of functional genetic circuits that respond to specific target ligands using RNA-based devices, specifically riboswitches, which induce downstream gene expression as a function of target ligand concentration.  Here, previously designed RNA sequences that bind to two small molecule ligands, p-aminophenylalanine (pAF) and theophylline (THP) were used as starting material. These sequences, termed aptamers, were cloned upstream of 15 nucleotide random sequences driving expression of a tetracycline resistance (tetA) readout gene.  In a subset of these random sequences presence of the ligand causes a structural change in the aptamer such that the ribosome binding site of tetA is available for binding.  Duplicate cultures of such aptamer/switch libraries were grown either in the presence or absence of the ligand (pAF, or THP).  Functional aptamer/switch pairs, riboswitches, were therefore enriched only in cultures containing the ligand.  Sequencing of the resulting cultures identified several hundred switch sequences that were > 2-fold more abundant in response to the presence or absence of the ligand.  Such changes in abundance represent putative successful riboswitches.  These studies are currently being expanded and refined so that enrichment and selection of functional riboswitches can take place within a single community of continuously cultured bacteria.  Additional experiments have been aimed at increasing the modularity if riboswitches through the inclusion of fusion proteins containing the gene the riboswitch was originally evolved with.  This has resulted in a fusion riboswitch evolved to induce expression of beta-galactosidase modified into a riboswitch designed to induce the expression of tetA.  This approach of rapidly and efficiently screening putative riboswitches, as well as increasing their modularity, has several applications as aptamers for new ligands are developed and synthetic genetic circuits become cornerstones of engineered microbial consortia of bio-industrial importance.