PCR on yeast colonies: An improved method for glyco-engineered Saccharomyces cerevisiae

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Abstract

Background: Saccharomyces cerevisiae is extensively used in bio-industries. However, its genetic engineering to introduce new metabolism pathways can cause unexpected phenotypic alterations. For example, humanisation of the glycosylation pathways is a high priority pharmaceutical industry goal for production of therapeutic glycoproteins in yeast. Genomic modifications can lead to several described physiological changes: biomass yields decrease, temperature sensitivity or cell wall structure modifications. We have observed that deletion of several N-mannosyltransferases in Saccharomyces cerevisiae, results in strains that can no longer be analyzed by classical PCR on yeast colonies. Findings. In order to validate our glyco-engineered Saccharomyces cerevisiae strains, we developed a new protocol to carry out PCR directly on genetically modified yeast colonies. A liquid culture phase, combined with the use of a Hot Start DNA polymerase, allows a 3-fold improvement of PCR efficiency. The results obtained are repeatable and independent of the targeted sequence; as such the protocol is well adapted for intensive screening applications. Conclusions: The developed protocol enables by-passing of many of the difficulties associated with PCR caused by phenotypic modifications brought about by humanisation of the glycosylation in yeast and allows rapid validation of glyco-engineered Saccharomyces cerevisiae cells. It has the potential to be extended to other yeast strains presenting cell wall structure modifications. © 2013 Bonnet et al.; licensee BioMed Central Ltd.

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Bonnet, C., Rigaud, C., Chanteclaire, E., Blandais, C., Tassy-Freches, E., Arico, C., & Javaud, C. (2013). PCR on yeast colonies: An improved method for glyco-engineered Saccharomyces cerevisiae. BMC Research Notes, 6(1). https://doi.org/10.1186/1756-0500-6-201

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