Development of an online reporter system to follow plasmid stability in Saccharomyces cerevisiae

Abstract

This work aims to develop a reporter system to follow plasmid stability online in Saccharomyces cerevisiae, which is widely used in industrial settings. The idea behind this is that a chromosomally integrated signaling protein is controlled by a repressor encoded on a plasmid. As soon as the plasmid is absent, the concentration of the repressor is reduced and the signaling protein can be expressed. Following preliminary tests, the PGK1 promoter was improved by insertion of lambda phage based operator sites in three locations in order to control yEGFP3 expression. Here, it could be recognized that a singular insertion of up to 48 bp in most cases does not interfere with PGK1 promoter activity to any significant extent. The combination with a highly expressed wild type lambda phage repressor in most promoter variants lead to a decreased expression of yEGFP3 up to 100µ20whereas in some combinations with repressors containing a NLS even an increase of the expression has been observed. The developed combinations with the highest repression of the reporter protein expression were able to detect a plasmid loss of 50R0or 30R0of the total cells, depending on the detection method.