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Nat Protoc. 2014 Apr;9(4):743-50. doi: 10.1038/nprot.2014.045. Epub 2014 Mar 6.

Transferring whole genomes from bacteria to yeast spheroplasts using entire bacterial cells to reduce DNA shearing.

Author information

1
Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, La Jolla, California, USA.
2
Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, Rockville, Maryland, USA.
3
1] Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, La Jolla, California, USA. [2] Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, Rockville, Maryland, USA.

Abstract

Direct cell-to-cell transfer of genomes from bacteria to yeast facilitates genome engineering for bacteria that are not amenable to genetic manipulation by allowing instead for the utilization of the powerful yeast genetic tools. Here we describe a protocol for transferring whole genomes from bacterial cells to yeast spheroplasts without any DNA purification process. The method is dependent on the treatment of the bacterial and yeast cellular mixture with PEG, which induces cell fusion, engulfment, aggregation or lysis. Over 80% of the bacterial genomes transferred in this way are complete, on the basis of structural and functional tests. Excluding the time required for preparing starting cultures and for incubating cells to form final colonies, the protocol can be completed in 3 h.

PMID:
24603933
DOI:
10.1038/nprot.2014.045
[Indexed for MEDLINE]

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