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


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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  1. Wang, H.H. et al. Genome-scale promoter engineering by coselection MAGE. Nat. Methods 9, 591–593 (2012).

    Article  Google Scholar 

  2. Noskov, V.N., Segall-Shapiro, T.H. & Chuang, R.Y. Tandem repeat coupled with endonuclease cleavage (TREC): a seamless modification tool for genome engineering in yeast. Nucleic Acids Res. 38, 2570–2576 (2010).

    Article  CAS  Google Scholar 

  3. Gibson, D.G. et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52–56 (2010).

    Article  CAS  Google Scholar 

  4. Itaya, M., Tsuge, K., Koizumi, M. & Fujita, K. Combining two genomes in one cell: stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome. Proc. Natl. Acad. Sci. USA 102, 15971–15976 (2005).

    Article  CAS  Google Scholar 

  5. Benders, G.A. et al. Cloning whole bacterial genomes in yeast. Nucleic Acids Res. 38, 2558–2569 (2010).

    Article  CAS  Google Scholar 

  6. Karas, B.J., Tagwerker, C., Yonemoto, I.T., Hutchison, C.A. III & Smith, H.O. Cloning the Acholeplasma laidlawii PG-8A genome in Saccharomyces cerevisiae as a yeast centromeric plasmid. ACS Syn. Biol. 1, 22–28 (2012).

    Article  CAS  Google Scholar 

  7. Tagwerker, C. et al. Sequence analysis of a complete 1.66 Mb Prochlorococcus marinus MED4 genome cloned in yeast. Nucleic Acids Res. 40, 10375–10383 (2012).

    Article  CAS  Google Scholar 

  8. Karas, B.J. et al. Direct transfer of whole genomes from bacteria to yeast. Nat. Methods 10, 410–412 (2013).

    Article  CAS  Google Scholar 

  9. Guerra-Tschuschke, I., Martin, I. & Gonzalez, M.T. Polyethylene glycol-induced internalization of bacteria into fungal protoplasts: electron microscopic study and optimization of experimental conditions. Appl. Environ. Microbiol. 57, 1516–1522 (1991).

    Article  CAS  Google Scholar 

  10. van Solingen, P. & van der Plaat, J.B. Fusion of yeast spheroplasts. J. Bacteriol. 130, 946–947 (1977).

    Article  CAS  Google Scholar 

  11. Markie, D. A simple assay for optimizing yeast-mammalian cell fusion conditions. Mol. Biotechnol. 6, 99–104 (1996).

    Article  CAS  Google Scholar 

  12. Dai, M., Ziesman, S., Ratcliffe, T., Gill, R.T. & Copley, S.D. Visualization of protoplast fusion and quantitation of recombination in fused protoplasts of auxotrophic strains of Escherichia coli. Metab. Eng. 7, 45–52 (2005).

    Article  CAS  Google Scholar 

  13. Tarshis, M., Salman, M. & Rottem, S. Fusion of mycoplasmas: the formation of cell hybrids. FEMS Microbiol. Lett. 66, 67–71 (1991).

    Article  CAS  Google Scholar 

  14. Akamatsu, T. & Sekiguchi, J. Selection methods in bacilli for recombinants and transformants of intra- and interspecific fused protoplasts. Arch. Microbiol. 134, 303–308 (1983).

    Article  CAS  Google Scholar 

  15. Kao, K.N. & Michayluk, M.R. A method for high-frequency intergeneric fusion of plant protoplasts. Planta 115, 13 (1974).

    Article  Google Scholar 

  16. Schaffner, W. Direct transfer of cloned genes from bacteria to mammalian cells. Proc. Natl. Acad. Sci. USA 77, 2163–2167 (1980).

    Article  CAS  Google Scholar 

  17. Maehara, T., Itaya, M., Ogura, M. & Tanaka, T. Effect of Bacillus subtilis BsuM restriction-modification on plasmid transfer by polyethylene glycol-induced protoplast fusion. FEMS Microbiol. Lett. 325, 49–55 (2011).

    Article  CAS  Google Scholar 

  18. Baigori, M., Sesma, F., de Ruiz Holgado, A.P. & de Mendoza, D. Transfer of plasmids between Bacillus subtilis and Streptococcus lactis. Appl. Environ. Microbiol. 54, 1309–1311 (1988).

    Article  CAS  Google Scholar 

  19. Gyuris, J. & Duda, E.G. High-efficiency transformation of Saccharomyces cerevisiae cells by bacterial minicell protoplast fusion. Mol. Cell Biol. 6, 3295–3297 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Li, L. & Blankenstein, T. Generation of transgenic mice with megabase-sized human yeast artificial chromosomes by yeast spheroplast-embryonic stem cell fusion. Nat. Protoc. 8, 1567–1582 (2013).

    Article  Google Scholar 

  21. Fleischmann, R.D. et al. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269, 496–512 (1995).

    Article  CAS  Google Scholar 

  22. Noskov, V.N. et al. Assembly of large, high G+C bacterial DNA fragments in yeast. ACS Syn. Biol. 1, 267–273 (2012).

    Article  CAS  Google Scholar 

  23. Hutchison, C.A. et al. Global transposon mutagenesis and a minimal Mycoplasma genome. Science 286, 2165–2169 (1999).

    Article  CAS  Google Scholar 

  24. Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  Google Scholar 

  25. Smith, H.O., Tomb, J.F., Dougherty, B.A., Fleischmann, R.D. & Venter, J.C. Frequency and distribution of DNA uptake signal sequences in the Haemophilus influenzae Rd genome. Science 269, 538–540 (1995).

    Article  CAS  Google Scholar 

  26. Kouprina, N. & Larionov, V. Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae. Nat. Protoc. 3, 371–377 (2008).

    Article  CAS  Google Scholar 

  27. Karas, B.J. et al. Assembly of eukaryotic algal chromosomes in yeast. J. Biol. Eng. 7, 30 (2013).

    Article  Google Scholar 

  28. Lartigue, C. et al. Genome transplantation in bacteria: changing one species to another. Science 317, 632–638 (2007).

    Article  CAS  Google Scholar 

  29. Lartigue, C. et al. Creating bacterial strains from genomes that have been cloned and engineered in yeast. Science 325, 1693–1696 (2009).

    Article  CAS  Google Scholar 

  30. Larionov, V., Kouprina, N., Solomon, G., Barrett, J.C. & Resnick, M.A. Direct isolation of human BRCA2 gene by transformation-associated recombination in yeast. Proc. Natl. Acad. Sci. USA 94, 7384–7387 (1997).

    Article  CAS  Google Scholar 

  31. Replogle, K., Hovland, L. & Rivier, D.H. Designer deletion and prototrophic strains derived from Saccharomyces cerevisiae strain W303-1a. Yeast 15, 1141–1149 (1999).

    Article  CAS  Google Scholar 

  32. Tully, J.G., Whitcomb, R.F., Clark, H.F. & Williamson, D.L. Pathogenic mycoplasmas: cultivation and vertebrate pathogenicity of a new spiroplasma. Science 195, 892–894 (1977).

    Article  CAS  Google Scholar 

  33. Poje, G. & Redfield, R.J. General methods for culturing Haemophilus influenzae. Methods Mol. Med. 71, 51–56 (2003).

    PubMed  Google Scholar 

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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



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).

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