Engineering Gram-Negative Microbial Cell Factories Using Transposon Vectors

  • Esteban Martínez-García
  • Tomás Aparicio
  • Víctor de Lorenzo
  • Pablo I. NikelEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1498)


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.

Key words

Mini-transposon Tn5 transposon Pseudomonas putida Escherichia coli Synthetic biology Metabolic engineering Microbial cell factory Genome editing 



The work described in this protocol was supported by the CAMBIOS Project of the Spanish Ministry of Economy and Competitiveness (RTC-2014-1777-3), the ST-FLOW (FP7-KBBE-2011-5-289326), EVOPROG (FP7-ICT-610730), ARISYS (ERC-2012-ADG-322797), and EmPowerPutida (EU-H2020-BIOTEC-2014-2015-6335536) Contracts of the European Union, and the PROMPT Project of the Autonomous Community of Madrid (CAM-S2010/BMD-2414). The authors declare that there is no conflict of interest. All the bacterial strains and plasmids described in the text are available upon request.


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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Esteban Martínez-García
    • 1
  • Tomás Aparicio
    • 1
  • Víctor de Lorenzo
    • 1
  • Pablo I. Nikel
    • 1
    Email author
  1. 1.Systems and Synthetic Biology ProgramCentro Nacional de Biotecnología (CNB-CSIC)MadridSpain

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