Figure 5

Introduction of newly developed vectors for heterologous gene expression. (a) Transformants that emerged after introduction of linearized plasmids developed in this study. Strains AM595 (h^– leu1-32 lys1-K24) and AM597 (h^– leu1-32 arg3-R25) were transformed with either pCLys1 or pCArg3 with or without NotI pretreatment and plated onto SD selection medium lacking lysine and arginine. (b) Expression of GFP introduced using pCLys1 and pCArg3. Transformants that emerged after introduction of linearized pCLys1-GFP and pCArg3-GFP into the lys1-K24 or arg3-R25 strains were detected and GFP fluorescence was monitored under fluorescence microscopy. (c) Confirmation of chromosomal integration by PCR. Integration of pCLys1-GFP or pCArg3-GFP into the chromosome was analyzed by PCR using primers indicated by arrowheads. Transformants #1 and #2 were strains in which GFP fluorescence was observed, whereas transformant #3 had no fluorescence. Lambda DNA digested with EcoT14I was used as a size marker. (d) Simultaneous expression of three proteins in a cell. Cells transformed with pCLys1, pCArg3, and pDUAL expressing uvi15-CFP, gpi16-YFP, and gar2-mCherry, respectively, were observed under fluorescence microscopy. Scale bars indicate 10 μm.