Abstract
A good part of the contemporary synthetic biology agenda aims at reprogramming microorganisms to enhance existing functions and/or perform new tasks. Moreover, the functioning of complex regulatory networks, or even a single gene, is revealed only when perturbations are entered in the corresponding dynamic systems and the outcome monitored. These endeavors rely on the availability of genetic tools to successfully modify á la carte the chromosome of target bacteria. Key aspects to this end include the removal of undesired genomic segments, systems for the production of directed mutants and allelic replacements, random mutant libraries to discover new functions, and means to stably implant larger genetic networks into the genome of specific hosts. The list of gram-negative species that are appealing for such genetic refactoring operations is growingly expanding. However, the repertoire of available molecular techniques to do so is very limited beyond Escherichia coli. In this chapter, utilization of novel tools is described (exemplified in two plasmids systems: pBAM1 and pEMG) tailored for facilitating chromosomal engineering procedures in a wide variety of gram-negative microorganisms.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
de Lorenzo V. (2010) Environmental biosafety in the age of Synthetic Biology: Do we really need a radical new approach? BioEssays 32: 926–931.
Gibson DG, Glass JI, Lartigue C et al (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329: 52–56.
Posfai G, Kolisnychenko V, Bereczki Z et al (1999) Markerless gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome. Nucleic Acids Res 27: 4409–4415.
Reznikoff WS. (2008) Transposon Tn5. Annu Rev Genet 42: 269–286.
Herrero M, de Lorenzo V, Timmis KN. (1990) Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in Gram-negative bacteria. J Bacteriol 172: 6557–6567.
de Lorenzo V, Herrero M, Jakubzik U et al (1990) Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gram-negative eubacteria. J Bacteriol 172: 6568–6572.
Kolter R, Inuzuka M, Helinski DR. (1978) Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Cell 15: 1199–1208.
Martínez-García E, Calles B, Arévalo-Rodríguez M et al (2011) pBAM1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes. BMC Microbiol 11:38.
Martinez-Garcia E, and de Lorenzo V. (2011)Engineering multiple genomic deletions in Gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. Environ Microbiol. DOI:10.1111/j.1462-2920.2011.02538.x.
Colleaux L, D’Auriol L, Galibert F et al (1988) Recognition and cleavage site of the intron-encoded omega transposase. Proc Natl Acad Sci USA 85: 6022–6026.
Wong SM, Mekalanos JJ. (2000) Genetic footprinting with mariner-based transposition in Pseudomonas aeruginosa. Proc Natl Acad Sci USA 97: 10191–10196.
Blatny JM, Brautaset T, Winther-Larsen HC et al (1997) Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in Gram-negative bacteria. Plasmid 38: 35–51.
Pratt LA, Kolter R. (1998) Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili. Mol Microbiol 30: 285–293.
Nelson KE, Weinel C, Paulsen IT et al (2002) Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4: 799–808.
Dos Santos VA, Heim S, Moore ER et al (2004) Insights into the genomic basis of niche specificity of Pseudomonas putida KT2440. Environ Microbiol 6: 1264–1286.
Choi KH, Kumar A, Schweizer HP. (2006) A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 64: 391–397.
Das S, Noe JC, Paik S et al (2005) An improved arbitrary primed PCR method for rapid characterization of transposon insertion sites. J Microbiol Methods 63: 89–94.
Horton RM, Hunt HD, Ho SN et al (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77: 61–68.
Sambrook J, Maniatis T, Fritsch EF (1989) Molecular cloning a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
de Lorenzo V, Timmis KN (1994) Analysis and construction of stable phenotypes in Gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol 235: 386–405.
Hanahan D. (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166: 557–580.
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor. New York.
Regenhardt D, Heuer H, Heim S et al. (2002) Pedi-gree and taxonomic credentials of Pseudomonas putida strain KT2440. Environ Microbiol 4: 912–915.
Fernandez S, de Lorenzo V, Perez-Martin J. (1995) Activation of the transcriptional regulator XylR of Pseudomonas putida by release of repression between functional domains. Mol Microbiol 16: 205–213.
Acknowledgments
Authors are indebted to S.M. Wong for the kind gift of pSW (I-SceI) plasmid. Development of the genetic tools described here was funded by generous grants of the CONSOLIDER program of the Spanish Ministry of Science and Innovation, by EU contracts BACSIN and MICROME and by Funds of the Autonomous Community of Madrid (TMEMS). All plasmids and strains described here are available upon request.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Martínez-García, E., de Lorenzo, V. (2012). Transposon-Based and Plasmid-Based Genetic Tools for Editing Genomes of Gram-Negative Bacteria. In: Weber, W., Fussenegger, M. (eds) Synthetic Gene Networks. Methods in Molecular Biology, vol 813. Humana Press. https://doi.org/10.1007/978-1-61779-412-4_16
Download citation
DOI: https://doi.org/10.1007/978-1-61779-412-4_16
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-411-7
Online ISBN: 978-1-61779-412-4
eBook Packages: Springer Protocols