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Construction of an Escherichia coli to Bacillus thuringiensis shuttle vector for large DNA fragments

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Abstract

Shuttle vectors for Bacillus thuringiensis or Bacillus cereus usually cannot hold fragments larger than 20 kb. With the development of genome research, shuttle vectors with higher loading capacity are necessary. We constructed an Escherichia coli to B. thuringiensis shuttle vector, pEMB0557, with a large loading capacity. This vector incorporated the ori60 replicon from B. thuringiensis subsp. kurstaki YBT-1520, erythromycin resistance (B. thuringiensis), and chloromycetin resistance (E. coli) genes. A bacterial artificial chromosome library of B. thuringiensis strain CT-43 was constructed and pEMB0557 was able to accommodate at least a 70-kb DNA fragment. Simultaneously, the cry1B gene on a 40-kb fragment could express a 140-kDa protein in plasmid-cured B. thuringiensis BMB171. Due to its high capacity and utility in expressing exogenous genes, pEMB0557 will be useful in cloning (especially silencing genes) and expressing large DNA fragments (e.g., gene clusters) in B. thuringiensis. Plasmid pEMB0557 provides a new tool for B. thuringiensis genome or B. cereus group research.

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Acknowledgments

This work was supported in part by the Hi-Tech Research and Development Project (863) of China (2006AA02Z174, 2006AA03A243), National Basic Research Program (973) of China (2009CB118902) and National Natural Science Foundation of China (30400003). We thank Dr. Meizhong Luo for kindly providing pBelloBAC11.

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  1. State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China

    Xiaoyan Liu, Donghai Peng, Yi Luo, Lifang Ruan, Ziniu Yu & Ming Sun

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  1. Xiaoyan Liu
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  2. Donghai Peng
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  3. Yi Luo
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  4. Lifang Ruan
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  5. Ziniu Yu
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  6. Ming Sun
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Correspondence to Ming Sun.

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Liu, X., Peng, D., Luo, Y. et al. Construction of an Escherichia coli to Bacillus thuringiensis shuttle vector for large DNA fragments. Appl Microbiol Biotechnol 82, 765–772 (2009). https://doi.org/10.1007/s00253-008-1854-y

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  • DOI: https://doi.org/10.1007/s00253-008-1854-y

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