Abstract
The Standard European Vector Architecture (SEVA) toolkit is a simple and powerful resource for constructing optimal plasmid vectors based on a backbone and three interchangeable modules flanked by uncommon restriction sites. Functional modules encode several origins of replication, diverse antibiotic selection markers, and a variety of cargoes with different applications. The backbone and DNA modules have been minimized and edited for flaws in their sequence and/or functionality. A protocol for the utilization of the SEVA platform to construct transcriptional and translational fusions between a promoter under study (the arsenic-responsive Pars of Pseudomonas putida KT2440) and the reporter lacZ gene is described. The resulting plasmid collection was instrumental to measure and compare the β-galactosidase activity that report gene expression (i.e., transcription and translation) in different genetic backgrounds.
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Acknowledgments
This work was supported by the BIO and FEDER CONSOLIDER-INGENIO programs of the Spanish Ministry of Economy and Competitiveness, the MICROME, ST-FLOW and ARISYS Contracts of the EU, the ERANET-IB Program, and funding from the Autonomous Community of Madrid (PROMPT).
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Durante-Rodríguez, G., de Lorenzo, V., Martínez-García, E. (2014). The Standard European Vector Architecture (SEVA) Plasmid Toolkit. In: Filloux, A., Ramos, JL. (eds) Pseudomonas Methods and Protocols. Methods in Molecular Biology, vol 1149. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-0473-0_36
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DOI: https://doi.org/10.1007/978-1-4939-0473-0_36
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