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

Nature Protocols volume 9, pages 743–750 (2014)Cite this article

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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.

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Figure 1: Diagram of direct cell-to-cell genome transfer from bacteria to yeast spheroplasts.
Figure 2: Timeline for tasks in the direct genome transfer protocol.

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Acknowledgements

This work was supported by Synthetic Genomics, Inc. B.J.K. was supported by the Natural Sciences and Engineering Research Council of Canada Postdoctoral Fellowships Program and by Synthetic Genomics, Inc. Y.S. was also supported by the US Defense Advanced Research Projects Agency contract no. N66001-12-C-4039 and the US Department of Energy cooperative agreement no. DE-EE0006109.

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Authors and Affiliations

  1. Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, La Jolla, California, USA

    Bogumil J Karas, Jelena Jablanovic, Edward Irvine, Lijie Sun, Philip D Weyman, Daniel G Gibson, J Craig Venter, Clyde A Hutchison III, Hamilton O Smith & Yo Suzuki

  2. Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, Rockville, Maryland, USA

    Li Ma, John I Glass & J Craig Venter

Authors
  1. Bogumil J Karas
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Contributions

B.J.K., J.J., P.D.W., D.G.G., J.I.G., J.C.V., C.A.H., H.O.S. and Y.S. designed the research. B.J.K., J.J., E.I., L.S., L.M., P.D.W., D.G.G., C.A.H. and Y.S. performed experiments. B.J.K., J.J., P.D.W. and Y.S. wrote the paper.

Corresponding authors

Correspondence to Bogumil J Karas or Yo Suzuki.

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Competing interests

J.C.V. is the Chief Executive Officer and Co-Chief Scientific Officer of Synthetic Genomics, Inc. (SGI). H.O.S. is the Co-Chief Scientific Officer and a member of the Board of Directors of SGI. C.A.H. is the Chairman of the SGI Scientific Advisory Board. D.G.G. is the Vice President for DNA Technology at SGI. All these four authors and the J. Craig Venter Institute hold SGI stock.

Supplementary information

Supplementary Note: Sequence of a yeast vector cassette.

Yeast elements that need to be inserted into bacterial genomes can be amplified from the plasmid pmycYAcTn (accession number GU593054)1. The sequence of CEN6 is underlined, that of the ARS is in italics, the HIS3 promoter is in bold, and the HIS3 coding sequence is in bold and underlined. (PDF 120 kb)

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Karas, B., Jablanovic, J., Irvine, E. et al. Transferring whole genomes from bacteria to yeast spheroplasts using entire bacterial cells to reduce DNA shearing. Nat Protoc 9, 743–750 (2014). https://doi.org/10.1038/nprot.2014.045

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