Efficient Design Strategy for Whole-Cell and Cell-Free Biosensors based on Engineered Riboswitches

Abstract

We have applied dual genetic selection to design a bacterial riboswitch with improved sensitivity by employing a high-affinity heterologous thiamine pyrophosphate (TPP) aptamer and modified selection conditions in Escherichia coli. The cells transformed with the engineered TPP riboswitches were characterized as whole-cell biosensors. The riboswitches were further studied in cell-free translation systems where they exhibited characteristics similar to those in vivo and a shorter response time for analysis. The flexibility of the riboswitch-based biosensors to accommodate different reporter genes was also demonstrated. Tuning of biosensor characteristics in vivo enables efficient development of aptamer-based whole-cell and cell-free biosensors.

Keywords:

• Cell-free biosensor
• dual genetic selection
• riboswitch
• whole-cell biosensor

This work was supported by the National Science Foundation (CCF0621523 and CCF0829536). We thank J. Kent Leach and Ram Rao for assistance with the luciferase assay.