Resumen de Design of standardized molecular tools to analyze regulatory properties and biotechnological applications of soil bacterium pseudomonas putida
The field of synthetic biology uses engineering principles to change or design de novo biological systems. Similar to engineering, synthetic biology employs standards to design and assemble genetic parts, devices and systems. The use of uniform molecular tools, which enables comparison of results obtained in different laboratories, requires adoption of common nomenclature and implements to develop platforms for data collection. Bacteria are an optimal system for reprogramming or re-implanting biological devices and Escherichia coli is a common reference strain for these tasks. Soil bacteria such as Pseudomonas putida are a valid alternative as a chassis because of their extreme adaptability to the external environment. P. putida KT2440, a strain derived from the wild type, toluene-degrading P. putida mt-2, is the most promising candidate for synthetic biology applications. Diverse genetic tools have been optimized for editing the P. putida KT2440 genome and include a collection of standardized vectors. This Thesis describes a number of molecular tools designed for work with P. putida KT2440 to clarify some questions regarding its regulation and use for biotechnological applications. We first designed and validated standardized plasmid- and transposon-based tools that allowed detailed analysis of the transcriptional state of given promoters in single cells and in clonal populations. Second, we explored the dynamics of the XylR-Pu regulatory network, which in P. putida is specialized in degrading aromatic compounds. We specifically studied single-cell responses of the promoter to increased production of its regulator and to different inducers. In addition, we analyzed XylR production in vivo after Crc protein-mediated repression. Finally, we showed the potential of heterologous expression systems for designing catalytic biofilms with P. putida KT2440.
