A Functional Cell-Based Genetic Screen for Variants of the B. subtilis pbuE Adenine Riboswitch that Bind Acyclovir and Theobromine

Alicea, Jesus

Undergraduate Honors Thesis

A Functional Cell-Based Genetic Screen for Variants of the B. subtilis pbuE Adenine Riboswitch that Bind Acyclovir and Theobromine Public Deposited

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Citations

Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/db78td75m

Abstract

Riboswitches are protein-independent RNA-based regulator elements capable of allowing or repressing expression of genes located downstream. Most riboswitch research has focused on characterization to determine crucial sequences and motifs involved in the structure and ligand-binding capabilities of riboswitches. These studies have revealed the capabilities of riboswitches, and a major goal in riboswitch research has been to engineer novel riboswitches capable of responding to non-natural ligands.

While previous studies have generated strategies to successfully engineer novel riboswitches, an efficient strategy has yet to be developed. In vitro strategies are capable of screening large libraries of both engineered riboswitches and small molecules. However, in vitro strategies lack selection for functional activity in cells, and many of the of the riboswitches developed in vitro don’t function in vivo. On the other hand, in vivo screening strategies have an intrinsic selection for functional activity in cells but are limited to smaller libraries amongst other constraints. A functional cell-based genetic screen for variants of the B. subtilis adenine riboswitch was employed against acyclovir and theobromine to find functional riboswitch variants while simultaneously probing this functional in vivo screening strategy. A feasibly sized library was created by varying five nucleotides in a region involved in ligand-binding. The screen did not produce any functional riboswitches, likely suggesting a larger library is necessary which requires a novel in vivo screening strategy.

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