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Construction of a thermo-sensitive pRI857 vector for efficient DNA capturing in Escherichia coli

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An Erratum to this article was published on 23 March 2017

Abstract

Objectives

To establish a positive cloning system with a zero background for high-throughput DNA cloning purpose.

Results

The cloning vector, pRI857, and the genomic-library construction vector, pRI857-BAC, were constructed based on the mechanism of expression of the thermo-sensitive cI857 repressor gene that can stringently repress the PR promoter and kanamycin resistance gene (PR-kan R) at 30 °C, but have no effect on PR-kan R gene at 37 °C or at higher temperatures. When the pRI857 vectors were transformed into E. coli with or without a target foreign DNA fragment inserted at the BfrBI site of the cI857 gene, only colonies with the foreign DNA fragment survive. We extended this method to construct a pRI857-BAC vector for genomic library cloning which displays an efficiency of ~107 cfu per µg of genomic DNA, with no empty vectors detected.

Conclusions

Cloning by indirect activation of resistance marker gene represents a novel DNA-capturing system, which can be widely applied for high-throughput DNA cloning.

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Acknowledgements

This work was supported by National Natural Science Foundation of China (Grant No. 21271104 and 21671103 for Yi J, and 31300111 for Wang Y). The strain Pseudomonas syrigae HS191 was a gift from Dr. Dennis C Gross (Texas A&M University).

Supporting information

Supplementary Table 1—The bacterial strains and plasmids used.

Supplementary Table 2—Sequence of primers used.

Supplementary Fig. 1—Plasmid map of the pRI857-BAC vector.

Supplementary Fig. 2—Cloning test of the ble R gene using pRI857 vector.

Supplementary Fig. 3—Cloning test of the PluxI-sfGFP gene using pRI857 vector.

Supplementary Fig. 4—Cloning test of the 16S rRNA gene from strain P. syringae HS191 using pRI857 vector.

Author information

Authors and Affiliations

  1. School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing, 210094, Jiangsu, China

    Kai Zhang, Huijuan Su, Muhan Yang, Jing Ge, Guiyao Li, Jun Yi & Yang Wang

  2. Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA

    Yang Wang

Authors
  1. Kai Zhang
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  2. Huijuan Su
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  3. Muhan Yang
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  4. Jing Ge
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  5. Guiyao Li
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  6. Jun Yi
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  7. Yang Wang
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Corresponding author

Correspondence to Yang Wang.

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Conflict of interest

The authors declare no conflict of interest.

Research involving human and animal rights

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Accession Numbers

The sequences reported in this article were deposited in the GenBank database [accession numbers: KX774470 (pRI857), KX774471 (pRI857-BAC)].

An erratum to this article is available at http://dx.doi.org/10.1007/s10529-017-2316-3.

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Zhang, K., Su, H., Yang, M. et al. Construction of a thermo-sensitive pRI857 vector for efficient DNA capturing in Escherichia coli . Biotechnol Lett 39, 905–909 (2017). https://doi.org/10.1007/s10529-017-2313-6

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  • DOI: https://doi.org/10.1007/s10529-017-2313-6

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