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




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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.



Autor: Ilaria Maria Benedetti, Victor de Lorenzo , Rafael Silva-Rocha

Fuente: http://plos.srce.hr/



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