Abstract
Here, we describe a bicistronic reporter system for the analysis of promoter activity in a variety of Gram-negative bacteria at both the population and single-cell levels. This synthetic genetic tool utilizes an artificial operon comprising the gfp and lacZ genes that are assembled in a suicide vector, which is integrated at specific sites within the chromosome of the target bacterium, thereby creating a monocopy reporter system. This tool was instrumental for the complete in vivo characterization of two promoters, Pb and Pc, that drive the expression of the benzoate and catechol degradation pathways, respectively, of the soil bacterium Pseudomonas putida KT2440. The parameterization of these promoters in a population (using β-galactosidase assays) and in single cells (using flow cytometry) was necessary to examine the basic numerical features of these systems, such as the basal and maximal levels and the induction kinetics in response to an inducer (benzoate). Remarkably, GFP afforded a view of the process at a much higher resolution compared with standard lacZ tests; changes in fluorescence faithfully reflected variations in the transcriptional regimes of individual bacteria. The broad host range of the vector/reporter platform is an asset for the characterization of promoters in different bacteria, thereby expanding the diversity of genomic chasses amenable to Synthetic Biology methods.
Highlights
The process of gene regulation in a living organism is vitally important for its adaptation to changing conditions in the environment
Construction of a Dual GFP-lacZ Reporter System Initially, we created a bicistronic system for promoter probing based on the expression of GFP and lacZ genes using as a reference a system for the single copy integration of a lacZ reporter that was previously described by Kessler and co-workers [36]
Here, we describe the implementation and validation of a dual bicistronic reporter system based on the GFP and lacZ genes
Summary
The process of gene regulation in a living organism is vitally important for its adaptation to changing conditions in the environment. The analysis of the complete genomes currently available reveals that a large amount of the genome encodes sequences related to gene regulation and transcription [1,2,3,4,5]. In the case of prokaryotes, the comparison between organisms with different lifestyles shows that the generalists (such as free living environmental bacteria) usually have a higher proportion of their genome content dedicated to gene regulation than the specialists (e.g., endosymbionts, [6]). The biochemical processes underlying gene regulation are driven by the collision of the reactants; the low amount of the reactants makes the system prone to a higher level of noise in the final output. Reactions endowed with low kinetic constants contribute to the increased noise of the cell [11]
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