GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.
Materials and methods:
SGA media:
The growth media (YPD) and selecting media (SD) used in this protocol is routinely used in yeast molecular biology. Please refer to Methods in Yeast (Amberg et al., 2005) for detailed descriptions.
LRK, LHRK media (for 400 ml total)
Add in order | Amount |
Yeast nitrogenous base with ammonium sulfate | 2.7 g |
Amino acid mix | 0.8 g |
Agar | 8 g |
dH2O | 360 mL |
Autoclave | |
20 % Dextrose | 40 ml |
Cool to 65˚C | |
100 mg/ml canavanine | 0.2 ml |
100 mg/ml thialysine | 0.2 ml |
Mix, pour 50 ml per plate |
LRK, LHRK + G418 media (for 400 ml total)
Add in order | Amount |
Yeast nitrogenous base without ammonium sulfate | 0.7 g |
Amino acid mix | 0.8 g |
Monosodium Glutamate | 0.4 |
Agar | 8 g |
dH2O | 360 mL |
Autoclave | |
20 % Dextrose | 40 ml |
Cool to 65˚C | |
100 mg/ml canavanine | 0.2 ml |
100 mg/ml thialysine | 0.2 ml |
200 mg/ml G418 | 0.4 ml |
Mix, pour 50 ml per plate |
Enriched sporulation media (for 400 ml total)
Add in order | Amount |
Potassium acetate | 4 g |
Yeast extract | 0.4 g |
Dextrose | 0.2 g |
Sporulation amino acid mix | 0.04 g |
Agar | 8 g |
dH2O | 360 mL |
Autoclave | |
Cool to 65˚C | |
200 mg/ml G418 | 0.4 ml |
Mix, pour 50 ml per plate |
This method can help efficiently incorporate a mitochondrial marker, Acp1-GFP into various mutant backgrounds. It relies on the use of a robotic system, and can easily adopted for use with any robotic system. This procedure can be also used for incorporating other types of markers. For example, to visualize ER, we routinely use the marker Erg11-GFP. In our representative images, a mutant and a wild type with the Acp1-GFP maker were visualiz ed by confocal microscopy to study the mitochondria morphology. The mutant showed disrupted mitochondrial phenotype, and is therefore a good candidate for further investigation.
This work was supported by the Biotechnology and Biological Sciences Research Council (grant 31/C15982), the Canadian Institutes of Health Research, the Canada Foundation for Innovation, the British Columbia Knowledge Development Fund, the Michael Smith Foundation for Health Research (grant to C.J.R. Loewen), and Fight For Sight.
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
SGA media | The growth media (YPD) and synthetic dropout media (SD) including LRK and LHRK, and enriched sporulation media used in this protocol is routinely used in yeast molecular biology. Please refer to Methods in Yeast (Amberg et al., 2005) for detailed descriptions. |