Testing mRPS12 TU

mRPS12 TU: mRPS12 TU was cloned into pSB416-GPD for expression in BY4741 Yeast and BY4741 mRPS12 TU knock out yeast. Competent mRPS12 + and – yeast strains were transformed with either pSB416-GPD with mRPS12 TU or empty pSB416-GPD and plated on CSM-Ura Plates. Colonies were allowed to grow for three days before they were picked and spotted onto two plates, one CSM-Ura with a fermentable Dextrose carbon source, and the other CSM-Ura but with a non-fermentable Glycerol carbon source

Testing mRPS12 TU

mls-yeGFP was cloned into pSB416-GPD for expression in BY4741 Yeast. Competent yeast cells were then transformed with either mls-yeGFP or empty pSB416 GPD and plated on CSM-Ura media to select for the presence of the pSB416-GPD plasmid. After three days of growth, colonies were picked and grown up in CSM-Ura liquid media for one day. Cultures were then fixed with paraformaldyhede for fluorescence microscopy. Using a fluorescence microscope, we compared brightfield and fluorescent views to determine if mls-yeGFP was causing transformed yeast to fluoresce. We then used a mitochondrial stain, Janus Green, to stain mitochondria in the mls-yeGFP cells and compared brightfield and fluorescent views to determine if the mls actually targeted the GFP protein to the mitochondria in the yeast cells.