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A standard for near-scarless plasmid construction using reusable DNA parts

Author

Listed:
  • Xiaoqiang Ma

    (National University of Singapore
    Singapore-MIT Alliance for Research and Technology)

  • Hong Liang

    (National University of Singapore
    Singapore-MIT Alliance for Research and Technology)

  • Xiaoyi Cui

    (National University of Singapore
    Singapore-MIT Alliance for Research and Technology)

  • Yurou Liu

    (National University of Singapore
    Singapore-MIT Alliance for Research and Technology)

  • Hongyuan Lu

    (National University of Singapore)

  • Wenbo Ning

    (National University of Singapore)

  • Nga Yu Poon

    (Singapore-MIT Alliance for Research and Technology)

  • Benjamin Ho

    (National University of Singapore)

  • Kang Zhou

    (National University of Singapore
    Singapore-MIT Alliance for Research and Technology)

Abstract

Here we report GT (Guanin/Thymine) standard (GTS) for plasmid construction under which DNA sequences are defined as two types of standard, reusable parts (fragment and barcode). We develop a technology that can efficiently add any two barcodes to two ends of any fragment without leaving scars in most cases. We can assemble up to seven such barcoded fragments into one plasmid by using one of the existing DNA assembly methods, including CLIVA, Gibson assembly, In-fusion cloning, and restriction enzyme-based methods. Plasmids constructed under GTS can be easily edited, and/or be further assembled into more complex plasmids by using standard DNA oligonucleotides (oligos). Based on 436 plasmids we constructed under GTS, the averaged accuracy of the workflow was 85.9%. GTS can also construct a library of plasmids from a set of fragments and barcodes combinatorically, which has been demonstrated to be useful for optimizing metabolic pathways.

Suggested Citation

  • Xiaoqiang Ma & Hong Liang & Xiaoyi Cui & Yurou Liu & Hongyuan Lu & Wenbo Ning & Nga Yu Poon & Benjamin Ho & Kang Zhou, 2019. "A standard for near-scarless plasmid construction using reusable DNA parts," Nature Communications, Nature, vol. 10(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-11263-0
    DOI: 10.1038/s41467-019-11263-0
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