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Folia Microbiologica

, Volume 58, Issue 5, pp 409–417 | Cite as

Development of a high-efficient transformation system of Bacillus pumilus strain DX01 to facilitate gene isolation via gfp-tagged insertional mutagenesis and visualize bacterial colonization of rice roots

  • Xinqian Shen
  • Yunpeng ChenEmail author
  • Tong Liu
  • Xiaolu Hu
  • Zhenfang Gu
Article

Abstract

A Tn5 transposition vector, pMOD-tet-egfp, was constructed and used for the random insertional mutagenesis of Bacillus pumilus. Various parameters were investigated to increase the transformation efficiency B. pumilus DX01 via Tn5 transposition complexes (transposome): bacterial growth phase, type of electroporation buffer, electric field strength, and recovery medium. Transformation efficiency was up to 3 × 104 transformants/μg of DNA under the optimized electroporation conditions, and a total of 1,467 gfp-tagged transformants were obtained. Fluorescence-activated cell sorting analysis showed that all gfp-tagged bacterial cells expressed GFP, indicating that foreign DNA has been successfully integrated into the genome of B. pumilus and expressed. Finally, flanking DNA sequences were isolated from several transformants and colonization of rice roots by B. pumilus DX01 was also studied. The method developed here will be useful for creating an insertion mutant library of gram-positive bacteria, thus facilitating their molecular genetic and cytological studies.

Keywords

Green Fluorescent Protein Rice Root Green Fluorescent Protein Expression Transformation Frequency Insertion Mutagenesis 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

This work has been supported by the National Natural Science Foundation of China (no. 31071721).

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Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2013

Authors and Affiliations

  • Xinqian Shen
    • 1
  • Yunpeng Chen
    • 1
    Email author
  • Tong Liu
    • 1
  • Xiaolu Hu
    • 1
  • Zhenfang Gu
    • 1
  1. 1.Department of Resources and Environmental Sciences, School of Agriculture and BiologyShanghai Jiaotong UniversityShanghaiPeople’s Republic of China

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