Ion-beam-induced gene transfer in Saccharomyces cerevisiae

Abstract

In this paper we present a novel method to induce gene transfer in yeast (Saccharomyces cerevisiae) using a low-energy ion beam. By accelerating nitrogen ions to the 50-60 keV energy range with ions fluences of 1-100 × 1015 ions/cm2, yeast cells were bombarded to characterize the decrease in survival with increasing energy level. Using bombardment conditions optimized for yeast cell survival (50 keV with ion fluences of 1 and 2 × 1015 ions/cm2), a compatible plasmid vector (pYES2) was successfully introduced into the yeast cell with higher concentrations of the plasmid providing improved plasmid transfer. To investigate expression of exotic genes in yeast, two marker genes - GFP and lipoic acid synthetase from Bacillus licheniformis - were chosen to subclone into pYES2 (designed pYGFP and pYlip respectively), and subsequently transformed into the cells. After 10 h of induction, the expression levels of these genes were analyzed. The pYGFP transformed yeast exhibited a high intensity of GFP protein in the yeast cells and the pYLip showed the expected additional protein-band at 34 kDa detected by SDS-polyacrylamide gel electrophoresis. As a method to transform yeast, low-energy ion beam bombardment is both highly efficient and since yeast can be transformed in less than 10 min, much more rapid than other yeast transformation methods. © 2009 Elsevier B.V. All rights reserved.