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.
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Acknowledgments
The authors would like to thank Dr. Stefan Werner and Dr. Ernst Weber for critical reading of this manuscript.
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© 2013 Springer Science+Business Media, New York
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Engler, C., Marillonnet, S. (2013). Combinatorial DNA Assembly Using Golden Gate Cloning. In: Polizzi, K., Kontoravdi, C. (eds) Synthetic Biology. Methods in Molecular Biology, vol 1073. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-625-2_12
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DOI: https://doi.org/10.1007/978-1-62703-625-2_12
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Publisher Name: Humana Press, Totowa, NJ
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