title: Engineering Gram-Negative Microbial Cell Factories Using Transposon Vectors.
abstract: The construction of microbial cell factories à la carte largely depends on specialized molecular biology and synthetic biology tools needed to reprogram bacteria for modifying their existing functions or for bestowing them with new-to-Nature tasks. In this chapter, we document the use of a series of broad-host-range mini-Tn5 vectors for the delivery of gene(s) into the chromosome of Gram-negative bacteria and for the generation of saturated, random mutagenesis libraries for studies of gene function. The application of these tailored mini-transposon vectors, which could also be used for chromosomal engineering of a wide variety of Gram-negative microorganisms, is demonstrated in the platform environmental bacterium Pseudomonas putida KT2440.
authors.affiliation: Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Calle Darwin, 3 Campus de Cantoblanco, 28049, Madrid, Spain.
authors.forename: Esteban
authors.initials: E
authors.lastname: Martínez-García
authors.affiliation: Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Calle Darwin, 3 Campus de Cantoblanco, 28049, Madrid, Spain.
authors.forename: Tomás
authors.initials: T
authors.lastname: Aparicio
authors.affiliation: Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Calle Darwin, 3 Campus de Cantoblanco, 28049, Madrid, Spain.
authors.forename: Víctor
authors.initials: V
authors.lastname: de Lorenzo
authors.affiliation: Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Calle Darwin, 3 Campus de Cantoblanco, 28049, Madrid, Spain. pablo.nikel@cnb.csic.es.
authors.forename: Pablo I
authors.initials: PI
authors.lastname: Nikel
mesh_terms: D060846:Cell Engineering / Q000379:methods; D002875:Chromosomes / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D006090:Gram-Negative Bacteria / Q000235:genetics*; D016296:Mutagenesis / Q000235:genetics; D016958:Pseudomonas putida / Q000235:genetics; D058615:Synthetic Biology / Q000379:methods
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial
keywords: Escherichia coli; Genome editing; Metabolic engineering; Microbial cell factory; Mini-transposon; Pseudomonas putida; Synthetic biology; Tn5 transposon