Quantitative, Non-Disruptive Monitoring of Transcription in Single Cells with a Broad-Host Range GFP-luxCDABE Dual Reporter System

Ilaria Maria Benedetti,Rafael Silva-Rocha,Victor De Lorenzo,Mark Isalan

doi:10.1371/journal.pone.0052000

Ilaria Maria Benedetti, Rafael Silva-Rocha + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0052000

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Journal: PloS one	Publication Date: Dec 28, 2012
Citations: 22	License type: CC BY 4.0

Affiliation: Centro Nacional de Biotecnología

Abstract

A dual promoter probe system based on a tandem bi-cistronic GFP-luxCDABE reporter cassette is described and implemented. This system is assembled in two synthetic, modular, broad-host range plasmids based on pBBR1 and RK2 origins of replication, allowing its utilization in an extensive number of gram-negative bacteria. We analyze the performance of this dual cassette in two hosts, Escherichia coli and Pseudomonas putida, by examining the induction properties of the lacIq-Ptrc expression system in the first host and the Pb promoter of the benzoate degradation pathway in the second host. By quantifying the bioluminescence signal produced through the expression of the lux genes, we explore the dynamic range of induction for the two systems (Ptrc-based and Pb-based) in response to the two inducers. In addition, by quantifying the fluorescence signals produced by GFP expression, we were able to monitor the single-cell expression profile and to explore stochasticity of the same two promoters by flow cytometry. The results provided here demonstrate the power of the dual GFP-luxCDABE cassette as a new, single-step tool to assess promoter properties at both the population and single-cell levels in gram-negative bacteria.

Highlights

Living organisms are equipped with complex machinery dedicated to interaction with the surrounding environment
The dual GFP-lux cassette system was assembled into two vectors, each harboring a kanamycin resistance marker and either a pBBR1- or RK2- based origin of replication, both of which are known to replicate in a wide number of hosts [23]
The resulting dual cassette is located downstream of an expanded multiple cloning site (MCS) composed of 12 unique restriction sites that are used for promoter cloning (Fig. 1b)

Summary

Introduction

Living organisms are equipped with complex machinery dedicated to interaction with the surrounding environment. The promoter sequence marks the physical region of the genome where both the expression apparatus (i.e., the RNA polymerase) and the regulatory elements (represented by transcriptional factors) are recruited in order to control the process of RNA production [2,3] In this sense, most of the signal integration process occurs at this specific spot through the interplay of a few to many elements (usually proteins), its final result being the control of the expression of the target gene [1,3,4]. The phenotype observed at the macroscopic scale using classical approaches (such as promoter analysis using enzymatic reporters e.g. b-galactosidase activity measurement) only represents the average behavior of the whole Such population-wide measurements of promoter activity say nothing on the performance of given promoters in individual cells, which can vary dramatically depending on the specific regulatory network [8,9,10,11]. Information on individual transcriptional activity and how it relates to the properties of the population as a whole is crucial for understanding the basic mechanisms underlying the gene expression process [8,9,11,12]

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