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pGP-B2E, a Recombinant Compatible TA/TB-Ligation Vector for Rapid and Inexpensive Gene Cloning

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Abstract

DNA cloning is the basic step for different fields of life science, and many efforts have been made to simplify this procedure. In this study, we report two general purpose plasmids (pGP), pGP-XB2E and pGP-B2E, for rapid and cost-effective construct of basic clones. The BciVI and XcmI cleavage sites are designed in pGP-XB2E to test plasmid linearization efficiency. The plasmid has better linearization efficiency by using BciVI which could almost completely digest 2 μg plasmid in 10 min with only one-tenth the recommended enzyme concentration. In order to further optimize the pGP-XB2E, a new plasmid, pGP-B2E, which removed XcmI cleavage site was designed. This vector is highly efficient for cloning PCR products up to 1812 bp, and the number of colonies was about five times that of pGP-XB2E. In addition to TA cloning, blunt-end PCR products with T ended in the primer could be positively linked to the T-vector pGP-B2E without A-tailing treatment (TB cloning). Moreover, as an entry vector, pGP-B2E was also compatible for gateway recombination reaction without frameshift mutations. In general, this vector provides a universal, quick, and cost-efficient method for basic molecular cloning.

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Acknowledgements

This work was supported by the National Key Research and Development Program of China (2016YFD0100601-15), Zhejiang Provincial Foundation for Natural Science (LZ16C130002, LY16C140005, LY17C130007), and Zhejiang Fundamental Public Welfare Research Program (LGN19C140008).

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Author notes

  1. Dongyue Li and Chao Zheng have contributed equally to this work.

Authors and Affiliations

  1. College of Plant Protection, Yunnan Agricultural University, Kunming, 650201, People’s Republic of China

    Dongyue Li, Weifang Liang & Jianping Chen

  2. State Key Laboratory of Breeding Base for Zhejiang Agricultural Products Quality and Safety, MOA Key Laboratory for Plant Protection and Biotechnology, Zhejiang Provincial Key Laboratory of Plant Virology, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, People’s Republic of China

    Dongyue Li, Chao Zheng, Jie Zhou, Bin Chen, Rumeng Xu, Wenxia Yuan, Ersong Zheng, Weifang Liang, Yong Yang, Lijuan He, Jianghua Shi, Xuming Wang & Jianping Chen

  3. College of Plant Protection, Northwest A&F University, Yangling, 712100, People’s Republic of China

    Chao Zheng

  4. College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua, 321004, People’s Republic of China

    Rumeng Xu, Wenxia Yuan, Ersong Zheng & Lijuan He

  5. Institute of Plant Virology, Ningbo University, Ningbo, People’s Republic of China

    Jianping Chen

  6. Institute of Biotechnology, Ningbo Academy of Agricultural Sciences, Ningbo, 315101, People’s Republic of China

    Chengqi Yan

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  1. Dongyue Li
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Correspondence to Xuming Wang or Jianping Chen.

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12033_2019_226_MOESM1_ESM.tif

Supplementary material 1 (TIFF 1436 kb). Fig. S1. Flowchart for constructing pGP-XB2E vector with sequence information. (A) The flowchart of constructing pGP-XB2E from pGP-X2E. First step, two original BciVI recognition sites in the pGP-X2E backbone were removed to generate an intermediate vector. Second step, two new BciVI sites near the XcmI sites were introduced. The new vector was named as pGP-XB2E. attL1 and attL2, recombination sites; BciVI and XcmI: restriction enzyme recognition sites; Cm(R), chloramphenicol resistance gene. (B) The sequence of original BciVI sites in the pGP-X2E backbone and sequence after mutation in the intermediate vector. (C) The sequence of XcmI sites and XcmI-BciVI sites after mutation

12033_2019_226_MOESM2_ESM.tif

Supplementary material 2 (TIFF 793 kb).Fig. S2. The cloning workflow of pGP-XB2E. pGP-XB2E could be digested with XcmI or BciVI to generate a vector with 3′ T overhangs for conventional TA cloning. Target fragments with 3′ A overhangs were amplified by Taq DNA polymerase. attL1 and attL2: Gateway recombination sites

12033_2019_226_MOESM3_ESM.tif

Supplementary material 3 (TIFF 14146 kb). Fig. S3. Ligation efficiency comparison for different sized inserts. (A) TA cloning efficiency of BciVI linearized pGP-XB2E and pGP-B2E with fragments of 495 bp, 1110 bp and 1812 bp in length. (B) TB cloning efficiency of pGP-B2E with the same fragments in blunt end. Photographs of colonies on plates were shown as representative of each ligation

12033_2019_226_MOESM4_ESM.tif

Supplementary material 4 (TIFF 7038 kb). Fig. S4. Construction and ligation efficiency test of pGP-B2ES. (A) Sequence of the cloning site of pGP-B2ES. The yellow part represents the PCR product. The pGP-B2ES vector has shorter sequences between attL site and PCR product which was mutated from pGP-B2E. (B) Ligation efficiency of BciVI linearized pGP-B2ES using DNA fragments of 495 bp, 1110 bp and 1812 bp. The molar ratio of fragment/vector was 10:1. The number of colonies were calculated and expressed as mean ± SD from three independent experiments. (C) Photographs of colonies on plates were shown as representative of each ligation

Supplementary material 5 (DOCX 18 kb)

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Li, D., Zheng, C., Zhou, J. et al. pGP-B2E, a Recombinant Compatible TA/TB-Ligation Vector for Rapid and Inexpensive Gene Cloning. Mol Biotechnol 62, 56–66 (2020). https://doi.org/10.1007/s12033-019-00226-x

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