Design and construction of a cellular biosensor for detection of autoinducer-2 quorum sensing inhibitors using a genetic toggle switch

Abstract

Bacteria employ molecular communication systems termed quorum sensing (QS) to sense cell density and organize collective behavior. Many of these behaviors have implications on modern society and human health. As the antimicrobial toolbox of classic antibiotics shrinks due to extensive spread of resistance in pathogenic bacteria, interfering with bacterial communication by ‘quenching’ the QS signals poses a promising strategy for novel antimicrobial drugs. Many bacterial biosensors have been used in the search of natural compounds interfering with QS. However, the majority of these detect compounds quenching autoinducer-1 QS and only a few detects compounds which quench other types of QS. Additionally, simple quorum quenching (QQ) biosensors are prone to bias and false-positive results. This thesis aims to employ synthetic biology tools to construct a modular and tunable cellular biosensor based on a bistable genetic circuit with two signal outputs for detecting compounds capable of quenching autoinducer-2 (AI-2) QS. In order to maintain tunability and modularity of the biosensor, the BASIC DNA Assembly system was used. A library of modular bioparts was generated and assembled into the biosensor plasmids. Escherichia coli DH5α, which is unable to produce AI-2, was used as primary sensor chassis. The promoter of the AI-2-regulated lsr-operon, Plsr, was employed as the sensing entity to control expression of a repressor, TetR, and a red fluorescent protein, mRFP1. The expression of sfGFP, a green fluorescent protein, was controlled by the TetR-repressed promoter Ptet. The biosensor plasmid therefore switches fluorescence color of DH5α depending on whether AI-2 QS is active or quenched. Different strategies were used to induce Plsr, but these proved unsuccessful. Leaky expression occurring through Plsr and lack of fluorescence by mRFP1 in the genetic construct were instead identified as possible reasons for the non-functional sensor. Further experiments revealed that one sensor module was functional, and that the inherent modularity of the BASIC DNA Assembly system allows for straightforward tuning of different parts. Future studies can therefore rely on the module containing Ptet and sfGFP and focus on tuning the Plsr- module.