Combinatorial DNA Assembly Using Golden Gate Cloning

  • Carola Engler
  • Sylvestre Marillonnet
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1073)

Abstract

A basic requirement for synthetic biology is the availability of efficient DNA assembly methods. We have previously reported the development of Golden Gate cloning, a method that allows parallel assembly of multiple DNA fragments in a one-tube reaction. Golden Gate cloning can be used for different levels of construct assembly: from gene fragments to complete gene coding sequences, from basic genetic elements to full transcription units, and finally from transcription units to multigene constructs. We provide here a protocol for DNA assembly using Golden Gate cloning, taking as an example the level of assembly of gene fragments to complete coding sequences, a level of cloning that can be used to perform DNA shuffling. Such protocol requires the following steps: (1) selecting fusion sites within parental sequences (sites at which parental sequences will be recombined), (2) amplifying all DNA fragments by PCR to add flanking restriction sites, (3) cloning the amplified fragments in intermediate constructs, and (4) assembling all or selected sets of intermediate constructs in a compatible recipient vector using a one-pot restriction-ligation.

Key words

Synthetic biology DNA assembly DNA shuffling Combinatorial Hierarchical Type IIS restriction enzymes Seamless cloning Modular cloning 
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Notes

Acknowledgments

The authors would like to thank Dr. Stefan Werner and Dr. Ernst Weber for critical reading of this manuscript.

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Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Carola Engler
    • 1
  • Sylvestre Marillonnet
    • 2
  1. 1.NOMAD BIOSCIENCE GMBHHalle (Saale)Germany
  2. 2.Department of Cell and Metabolic BiologyLeibniz-Institut für PflanzenbiochemieHalleGermany

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