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Author Notes:

imatsum@emory.edu

Ichiro Matsumura conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

The author declares that he has no competing interests.

Subjects:

Research Funding:

This work was supported by the National Science Foundation (MCB 1359575, MCB 1413062).

Keywords:

  • Science & Technology
  • Multidisciplinary Sciences
  • Science & Technology - Other Topics
  • BioBrick assembly
  • Methylase-assisted cloning
  • Synthetic biology
  • Laboratory automation
  • DNA methyltransferase
  • RESTRICTION
  • CLONING
  • EFFICIENCY
  • STANDARDS
  • PROMOTER

Methylase-assisted subcloning for high throughput BioBrick assembly

Tools:

Journal Title:

PEERJ

Volume:

Volume 8

Publisher:

, Pages e9841-e9841

Type of Work:

Article | Final Publisher PDF

Abstract:

The BioBrick standard makes possible iterated pairwise assembly of cloned parts without any depletion of unique restriction sites. Every part that conforms to the standard is compatible with every other part, thereby fostering a worldwide user community. The assembly methods, however, are labor intensive or inefficient compared to some newer ones so the standard may be falling out of favor. An easier way to assemble BioBricks is described herein. Plasmids encoding BioBrick parts are purified from Escherichia coli cells that express a foreign site-specific DNA methyltransferase, so that each is subsequently protected in vitro from the activity of a particular restriction endonuclease. Each plasmid is double-digested and all resulting restriction fragments are ligated together without gel purification. The ligation products are subsequently double-digested with another pair of restriction endonucleases so only the desired insert-recipient vector construct retains the capacity to transform E. coli. This 4R/2M BioBrick assembly protocol is more efficient and accurate than established workflows including 3A assembly. It is also much easier than gel purification to miniaturize, automate and perform more assembly reactions in parallel. As such, it should streamline DNA assembly for the existing community of BioBrick users, and possibly encourage others to join.

Copyright information:

©2020 Matsumura

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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