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Related Experiment Videos

New plasmid vector and host system for genetic engineering in Bacillus sphaericus.

M Qiao1, C Gao, B Cai

  • 1Institute for Molecular Biology, Nankai University Tianjin, People's Republic of China.

Plasmid
|May 1, 1992
PubMed
Summary

A new plasmid, pNQ116, enables high-level gene expression in Bacillus sphaericus. This stable plasmid vector does not affect the mosquito larvicidal activity of the bacterium.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biotechnology

Background:

  • Bacillus sphaericus is a bacterium with significant applications, including mosquito larvicidal activity.
  • Development of efficient plasmid vectors is crucial for genetic manipulation and enhancing the capabilities of Bacillus sphaericus.
  • Existing vectors may have limitations in gene expression levels or host range.

Purpose of the Study:

  • To construct and characterize a novel plasmid vector, pNQ116, for use in Bacillus sphaericus.
  • To evaluate the gene expression capabilities and stability of the new plasmid in various Bacillus sphaericus strains.
  • To assess the impact of the plasmid on the native mosquito larvicidal activity of Bacillus sphaericus.

Main Methods:

  • Construction of pNQ116 by cloning a promoter fragment from B. sphaericus Ts-1 into pNQ112.

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  • Characterization of plasmid features, including antibiotic resistance markers (chloramphenicol and kanamycin) and a restriction endonuclease polylinker.
  • Transformation of pNQ116 into multiple Bacillus sphaericus strains (AS 1.270, AS 1.465, AS 1.469, and 2362) and assessment of transformation frequencies.
  • Evaluation of plasmid stability under nonselective conditions.
  • Testing the effect of pNQ116 presence on the mosquito larvicidal activity of B. sphaericus 2362.

    • Main Results:

    • The novel plasmid pNQ116 (5.23 kb) was successfully constructed, conferring chloramphenicol and kanamycin resistance.
    • The Ts-1 promoter drives high-level expression of the cat-86 gene on pNQ116.
    • Efficient transformation of pNQ116 into B. sphaericus strains was achieved, with frequencies ranging from 10^2 to 10^3 transformants per microgram of DNA.
    • The plasmid demonstrated stable maintenance in host strains without selective pressure.
    • Introduction of pNQ116 into B. sphaericus 2362 did not impair its mosquito larvicidal efficacy.

    Conclusions:

    • The plasmid pNQ116 is a functional and stable expression vector for Bacillus sphaericus.
    • High gene expression levels can be achieved using the Ts-1 promoter within this vector system.
    • pNQ116 represents a valuable tool for genetic engineering of Bacillus sphaericus without compromising its beneficial larvicidal properties.