A Fast and Practical Yeast Transformation Method Mediated by Escherichia coli Based on a Trans-Kingdom Conjugal Transfer System: Just Mix Two Cultures and Wait One Hour

PLoS One. 2016 Feb 5;11(2):e0148989. doi: 10.1371/journal.pone.0148989. eCollection 2016.

Abstract

Trans-kingdom conjugation is a phenomenon by which DNA is transferred into a eukaryotic cell by a bacterial conjugal transfer system. Improvement in this method to facilitate the rapid co-cultivation of donor bacterial and recipient eukaryotic cell cultures could make it the simplest transformation method, requiring neither isolation of vector DNA nor preparation of competent recipient cells. To evaluate this potential advantage of trans-kingdom conjugation, we examined this simple transformation method using vector combinations, helper plasmids, and recipient Saccharomyces cerevisiae strains. Mixing donor Escherichia coli and recipient S. cerevisiae overnight cultures (50 μL each) consistently yielded on the order of 10(1) transformants using the popular experimental strain BY4742 derived from S288c and a shuttle vector for trans-kingdom conjugation. Transformation efficiency increased to the order of 10(2) using a high receptivity trans-kingdom conjugation strain. In addition, either increasing the amount of donor cells or pretreating the recipient cells with thiols such as dithiothreitol improved the transformation efficiency by one order of magnitude. This simple trans-kingdom conjugation-mediated transformation method could be used as a practical yeast transformation method upon enrichment of available vectors and donor E. coli strains.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Bacterial / genetics*
  • Escherichia coli / genetics*
  • Gene Transfer Techniques*
  • Genetic Vectors*
  • Saccharomyces cerevisiae / genetics*

Substances

  • DNA, Bacterial

Grant support

This work was supported in part by JSPS KAKENHI (21510209 and 25660007). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.