Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes.

We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by sim...

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Autores principales:	Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2009
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/1ed45db2e77448e0907590df9cc03165
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spelling	oai:doaj.org-article:1ed45db2e77448e0907590df9cc031652021-11-25T06:22:42ZGolden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes.1932-620310.1371/journal.pone.0005553https://doaj.org/article/1ed45db2e77448e0907590df9cc031652009-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/19436741/?tool=EBIhttps://doaj.org/toc/1932-6203We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call 'Golden Gate shuffling', is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.Carola EnglerRamona GruetznerRomy KandziaSylvestre MarillonnetPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 4, Iss 5, p e5553 (2009)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Carola Engler Ramona Gruetzner Romy Kandzia Sylvestre Marillonnet Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes.
description	We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call 'Golden Gate shuffling', is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.
format	article
author	Carola Engler Ramona Gruetzner Romy Kandzia Sylvestre Marillonnet
author_facet	Carola Engler Ramona Gruetzner Romy Kandzia Sylvestre Marillonnet
author_sort	Carola Engler
title	Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes.
title_short	Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes.
title_full	Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes.
title_fullStr	Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes.
title_full_unstemmed	Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes.
title_sort	golden gate shuffling: a one-pot dna shuffling method based on type iis restriction enzymes.
publisher	Public Library of Science (PLoS)
publishDate	2009
url	https://doaj.org/article/1ed45db2e77448e0907590df9cc03165
work_keys_str_mv	AT carolaengler goldengateshufflingaonepotdnashufflingmethodbasedontypeiisrestrictionenzymes AT ramonagruetzner goldengateshufflingaonepotdnashufflingmethodbasedontypeiisrestrictionenzymes AT romykandzia goldengateshufflingaonepotdnashufflingmethodbasedontypeiisrestrictionenzymes AT sylvestremarillonnet goldengateshufflingaonepotdnashufflingmethodbasedontypeiisrestrictionenzymes
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Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes.

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