Designer Sinorhizobium meliloti strains and multi-functional vectors for direct inter-kingdom transfer of high G+C content DNA

Brumwell, S. L.; MacLeod, M. R.; Huang, T.; Cochrane, R.; Meaney, R. S.; Zamani, M.; Matysiakiewicz, O.; Janakirama, P.; Edgell, D.; Charles, T. C.; Finan, T. M.; Karas, B. J.

doi:10.1101/449991

bioRxiv · 10.1101/449991

Designer Sinorhizobium meliloti strains and multi-functional vectors for direct inter-kingdom transfer of high G+C content DNA

Brumwell, S. L.·MacLeod, M. R.·Huang, T.·Cochrane, R.·Meaney, R. S.·Zamani, M.·Matysiakiewicz, O.·Janakirama, P.·Edgell, D.·Charles, T. C.·Finan, T. M.·Karas, B. J.

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Abstract

Storage and manipulation of large DNA fragments is crucial for synthetic biology applications, yet DNA with high G+C content can be unstable in many host organisms. Here, we report the development of Sinorhizobium meliloti as a new universal host that can store DNA, including high G+C content, and mobilize DNA to Escherichia coli, Saccharomyces cerevisiae, and the eukaryotic microalgae Phaeodactylum tricornutum. We deleted the S. meliloti hsdR restriction-system to enable DNA transformation with up to 1.4 x 105 efficiency. Multi-host and multi-functional shuttle vectors (MHS) were constructed and shown to stably replicate in S. meliloti, E. coli, S. cerevisiae, and P. tricornutum, with a copy-number inducible E. coli origin for isolating plasmid DNA. Crucially, we demonstrated that S. meliloti can act as a universal conjugative donor for MHS plasmids with a cargo of at least 62 kb of G+C rich DNA derived from Deinococcus radiodurans.

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Brumwell, S. L., MacLeod, M. R., Huang, T., Cochrane, R., Meaney, R. S., Zamani, M., Matysiakiewicz, O., Janakirama, P., Edgell, D., Charles, T. C., Finan, T. M., Karas, B. J.. 2018-10-22. Designer Sinorhizobium meliloti strains and multi-functional vectors for direct inter-kingdom transfer of high G+C content DNA. https://doi.org/10.1101/449991

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