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A Tn7-based broad-range bacterial cloning and expression system

Nature Methods volume 2, pages 443–448 (2005)Cite this article

Abstract

For many bacteria, cloning and expression systems are either scarce or nonexistent. We constructed several mini-Tn7 vectors and evaluated their potential as broad-range cloning and expression systems. In bacteria with a single chromosome, including Pseudomonas aeruginosa, Pseudomonas putida and Yersinia pestis, and in the presence of a helper plasmid encoding the site-specific transposition pathway, site- and orientation-specific Tn7 insertions occurred at a single attTn7 site downstream of the glmS gene. Burkholderia thailandensis contains two chromosomes, each containing a glmS gene and an attTn7 site. The Tn7 system allows engineering of diverse genetic traits into bacteria, as demonstrated by complementing a biofilm-growth defect of P. aeruginosa, establishing expression systems in P. aeruginosa and P. putida, and 'GFP-tagging' Y. pestis. This system will thus have widespread biomedical and environmental applications, especially in environments where plasmids and antibiotic selection are not feasible, namely in plant and animal models or biofilms.

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Figure 1: Suicide delivery vectors and mini-Tn7 elements.
Figure 2: Integration of mini-Tn7 in P. aeruginosa.
Figure 3: Tn7 insertion sites in various gram-negative bacteria.
Figure 4: Mini-Tn7 vector applications.

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Acknowledgements

H.P.S. was supported by National Institutes of Health grant AI058141. We thank K. Giesler and K. Quinn for their contributions to this project, and R. Gillis (University of Rochester) for assisting with biofilm experiments.

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Authors and Affiliations

  1. Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, 80523, Colorado, USA

    Kyoung-Hee Choi, Jared B Gaynor, Kimberly G White, Carolina Lopez, Catharine M Bosio, RoxAnn R Karkhoff-Schweizer & Herbert P Schweizer

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  1. Kyoung-Hee Choi
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  2. Jared B Gaynor
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Correspondence to Herbert P Schweizer.

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Supplementary information

Supplementary Fig. 1

Maps of mini-Tn7 suicide delivery base vectors. (PDF 112 kb)

Supplementary Fig. 2

Maps of selected mini-Tn7 elements. (PDF 150 kb)

Supplementary Fig. 3

Mini-Tn7 insertion does not affect GlmS expression. (PDF 93 kb)

Supplementary Fig. 4

Stability of mini-Tn7 insertions in the absence of selection. (PDF 156 kb)

Supplementary Table 1

Plasmid vectors derived in this study and GenBank accession numbers. (PDF 86 kb)

Supplementary Table 2

glmS genes in annotated bacterial genomes. (PDF 181 kb)

Supplementary Methods (PDF 161 kb)

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Choi, KH., Gaynor, J., White, K. et al. A Tn7-based broad-range bacterial cloning and expression system. Nat Methods 2, 443–448 (2005). https://doi.org/10.1038/nmeth765

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