Creating a Riboswitch-Based Whole-Cell Biosensor for BPA

Detection of small organic molecules using riboswitches in whole-cell biosensors could allow for screening of environmental pollutants, such as bisphenol A (BPA), in an effective and economical manner. Riboswitches are RNA sequences typically embedded within the 5â?? untranslated region of mRNA that are capable of regulating gene expression and, when paired with a reporter gene, can signal the presence of small organic
molecules. In order to create a riboswitch in Escherichia coli that could bind BPA and, upon binding, translationally upregulate the expression of gfp, we investigated (1) the best method of engineering a novel BPA-activated riboswitch via dual genetic selection and (2) the effectiveness of using such a riboswitch to detect BPA.
Dual genetic selection was performed using a tetA-gfp reporter downstream of a mutagenized thiM riboswitch. Two E. coli libraries â?? Library 1 (3 x 104 members) and Library 2 (4 x 106 members) â?? were subjected to positive selection followed by negative selection. Library 1 was only subjected to one round of selection, whereas Library 2 was subjected to several rounds of selection under increasingly stringent selection conditions. These libraries were then screened for BPA-activated riboswitches via a GFP assay. Pre- selection sequencing of random members revealed that Library 1 and Library 2 were both highly diverse; no conserved sequences were identified within each library. Initial
analysis of post-selection hits suggests that multiple rounds of selection may be necessary. While more investigations must be conducted to verify these preliminary results and to determine the effectiveness of using a riboswitch to detect BPA, this technology serves as a promising method for detecting pollutants in the environment.