Investigating the Utility of a Bacterial Auxotroph System for Novel Riboswitch Selection

Badger, Nickles

Undergraduate Honors Thesis

Investigating the Utility of a Bacterial Auxotroph System for Novel Riboswitch Selection Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/d791sh65v

Abstract

Riboswitches serve as metabolite-sensitive RNA regulatory elements in the leader sequences of many prokaryotic transcripts. Synthetic riboswitches selectively responsive to medically or industrially relevant small molecules have potential as useful mechanisms for dosage regulation and biosensing applications. Development of such novel functional RNAs has remained challenging due to large library sizes, throughput limitations of screening approaches, and unreliable in vivo function of riboswitches obtained via in vitro methods. To overcome these impediments, a bacterial double auxotroph selection system initially employed for transcription factor binding specificity determination was repurposed to investigate the utility of the approach for identifying novel riboswitches responsive to alternate ligands from large libraries (Meng & Wolfe, 2006). Library sequences used in the system possessed a riboswitch scaffold with randomized bases in the aptamer positioned upstream of positive and negative selectable markers, hisB and URA3 in a bicistronic reporter plasmid. In histidine drop-out medium, members with a riboswitch responsive to ligand were conferred a survival advantage by reduced attenuation in expression of hisB, an essential enzyme in histidine biosynthesis. Validation experiments using P4C, a known functional derivative of the purine responsive pbuE riboswitch, inserted upstream of the reporter genes, demonstrated ligand dependent survival and potential utility of the system for library selection. Furthermore, a P4C scaffolded library influencing reporter genes in a selection strain grown in solid drop-out media with optimized 3- aminotriazole inhibitor and 2-aminopurine ligand was subjected to positive selection. Results suggest library members which inhibit expression even in the presence of purine ligand were successfully selected against. Mutant library members and other constructs conferring a survival advantage and outcompeting properly constructed members despite the lack of a functional riboswitch remain a challenge. Approaches to mitigate against the introduction of these evaders were investigated and robust counterselection appears to be necessary to achieve successful implementation of an auxotroph-based riboswitch selection tool. Although continued efforts are necessary, the approach offers a potential avenue to select for therapeutically applicable biosensors responsive to alternate ligands from large libraries.

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