Fluoride Action Network

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Abstract

Biocontainment systems are needed to neutralize genetically modified organisms (GMOs) that pose ecological threats outside of controlled environments. In contrast, benign selection markers complement GMOs with reduced fitness. Benign selection agents serve as alternatives to antibiotics, which are costly and risk spread of antibiotic resistance. Here, we present a yeast biocontainment strategy leveraging engineered fluoride sensitivity and DNA vectors enabling use of fluoride as a selection agent. The biocontainment system addresses the scarcity of platforms available for yeast despite their prevalent use in industry and academia. In the absence of fluoride, the biocontainment strain exhibits phenotypes nearly identical to those of the wildtype strain. Low fluoride concentrations severely inhibit biocontainment strain growth, which is restored upon introduction of fluoride-based vectors. The biocontainment strategy is stringent, easily implemented, and applicable to several eukaryotes. Further, the DNA vectors enable genetic engineering at reduced costs and eliminate risks of propagating antibiotic resistance.

Fig. 1: Engineered fluoride sensitivity augments biocontainment and selection systems.

figure1

Yeast cells lacking native fluoride exporter genes (FEX1/FEX2) are highly sensitized to low concentrations of fluoride yet retain wildtype phenotypes in the absence of the ion. Accordingly, this mechanism befits biocontainment of genetically modified organisms (GMOs) that pose ecological risks outside of laboratory environments. Alternatively, fluoride sensitivity can be leveraged to provide an alternative selection marker in GMOs with reduced fitness and likelihood of persistence in the environment.

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*Original full-text study online at https://www.nature.com/articles/s41467-020-19271-1

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Acknowledgements

We thank Dr. Stephen Streatfield for helpful discussions regarding biocontainment systems and Professor Simon Avery for pSVA13. J.I.Y. acknowledges support from a National Science Foundation Graduate Research Fellowship under grant no. 1650114. The authors further acknowledge funding support from the National Science Foundation (MCB-1553721).

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Affiliations

  1. Department of Chemical Engineering, University of California Santa Barbara, Santa Barbara, CA, 93106, USA

    Justin I. Yoo, Susanna Seppälä & Michelle A. O?Malley

Contributions

J.I.Y. and M.A.O. conceived the study, designed the experiments, analyzed the data, and wrote the manuscript. J.I.Y. and S.S. conducted the experiments.

Corresponding author

Correspondence to Michelle A. O’Malley.

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Competing interests

The authors declare the following competing interests: J.I.Y. and M.A.O. are authors on a patent application, application number 63072933, which has been filed. All other authors declare no competing interests.

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Yoo, J.I., Seppälä, S. & O?Malley, M.A. Engineered fluoride sensitivity enables biocontainment and selection of genetically-modified yeasts. Nat Commun 11, 5459 (2020). https://doi.org/10.1038/s41467-020-19271-1

Nature Communications ISSN 2041-1723 (online)

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