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EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology

  • Hung-En Lai
  • Simon Moore
  • Karen Polizzi
  • Paul Freemont
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1772)

Abstract

Development of advanced synthetic biology tools is always in demand since they act as a platform technology to enable rapid prototyping of biological constructs in a high-throughput manner. EcoFlex is a modular cloning (MoClo) kit for Escherichia coli and is based on the Golden Gate principles, whereby Type IIS restriction enzymes (BsaI, BsmBI, BpiI) are used to construct modular genetic elements (biological parts) in a bottom-up approach. Here, we describe a collection of plasmids that stores various biological parts including promoters, RBSs, terminators, ORFs, and destination vectors, each encoding compatible overhangs allowing hierarchical assembly into single transcription units or a full-length polycistronic operon or biosynthetic pathway. A secondary module cloning site is also available for pathway optimization, in order to limit library size if necessary. Here, we show the utility of EcoFlex using the violacein biosynthesis pathway as an example.

Keywords

Synthetic biology Golden gate MoClo Violacein DNA assembly 

References

  1. 1.
    Kim E, Moore BS, Yoon YJ (2015) Reinvigorating natural product combinatorial biosynthesis with synthetic biology. Nat Chem Biol 11:649–660CrossRefGoogle Scholar
  2. 2.
    Temme K, Zhao D, Voigt CA (2012) Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca. Proc Natl Acad Sci U S A 109:2–7CrossRefGoogle Scholar
  3. 3.
    Moore SJ, Lai HE, Kelwick RJR et al (2016) EcoFlex: a multifunctional MoClo Kit for E. coli synthetic biology. ACS Synth Biol 5:1059–1069CrossRefGoogle Scholar
  4. 4.
    Engler C, Gruetzner R, Kandzia R et al (2009) Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4:e5553CrossRefGoogle Scholar
  5. 5.
    iGEM (2017) Registry of standard biological parts. http://parts.igem.org. Accessed 17 July 2017
  6. 6.
    Chen Y-J, Liu P, Nielsen A a K et al (2013) Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat Methods 10:659–664CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Hung-En Lai
    • 1
    • 2
  • Simon Moore
    • 1
    • 2
  • Karen Polizzi
    • 2
    • 3
  • Paul Freemont
    • 1
    • 2
  1. 1.Department of MedicineImperial College LondonLondonUK
  2. 2.Centre for Synthetic Biology and InnovationImperial College LondonLondonUK
  3. 3.Department of Life SciencesImperial College LondonLondonUK

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