Tn7-Based Device for Calibrated Heterologous Gene Expression in Pseudomonas putida

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• 作者：Sebastian Zobel ; Ilaria Benedetti ; Lara Eisenbach ; Victor de Lorenzo ; Nick Wierckx ; Lars M. Blank
• 刊名：ACS Synthetic Biology
• 出版年：2015
• 出版时间：December 18, 2015
• 年：2015
• 卷：4
• 期：12
• 页码：1341-1351
• 全文大小：569K
• ISSN：2161-5063

文摘

The soil bacterium Pseudomonas putida is increasingly attracting considerable interest as a platform for advanced metabolic engineering through synthetic biology approaches. However, genomic context, gene copy number, and transcription/translation interplay often introduce considerable uncertainty to the design of reliable genetic constructs. In this work, we have established a standardized heterologous expression device in which the promoter strength is the only variable; the remaining parameters of the flow have stable default values. To this end, we tailored a mini-Tn7 delivery transposon vector that inserts the constructs in a single genomic locus of P. putida鈥檚 chromosome. This was then merged with a promoter insertion site, an unvarying translational coupler, and a downstream location for placing the gene(s) of interest under fixed assembly rules. This arrangement was exploited to benchmark a collection of synthetic promoters with low transcriptional noise in this bacterial host. Growth experiments and flow cytometry with single-copy promoter鈥揋FP constructs revealed a robust, constitutive behavior of these promoters, whose strengths and properties could be faithfully compared. This standardized expression device significantly extends the repertoire of tools available for reliable metabolic engineering and other genetic enhancements of P. putida.