2.2.1 E. coli – media and growth
2.2.1.1Media for E. coli
LB-medium: 0.5% (w/v) yeast extract, 1% (w/v) bacto-tryptone, 1% (w/v) NaCl.
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Above mentioned composition of the media were used for preparing liquid cultures. For preparation of solid media (LB or LB-Amp plates) 2% (w/v) bacto-agar was added to the liquid media solutions and autoclaved (120ºC, 20 min). The ampicillin was added after the media had been cooled down to 50ºC.
2.2.1.2Cultivation of E. coli
Liquid medium (usually 50 ml of LB-Amp) was inoculated with the single colony from the plate and grown overnight at 37°C while shaking at 140 rpm. If necessary, cells were grown for up to 24h at lower temperatures (30 or 24°C).
2.2.2 S. cerevisiae – media and growth
2.2.2.1Media for S.cerevisiae Non-selective media:
YP-medium: 10 g yeast extract, 20 g bacto-peptone, H2O to 930 ml, pH 5.0 (adjusted with HCl).
YPD-medium: YP-medium supplemented with 2% glucose.
YPG-medium: YP-medium supplemented with 3% (v/v) glycerol.
YPGal-medium: YP-medium supplemented with 2% galactose.
Lactate medium: 3 g yeast extract, 1 g KH2PO4, 1 g NH4Cl, 0.5 g CaCl2 x 2H2O, 0.5 g NaCl, 1.1 g MgSO4 x 6H2O, 0.3 ml 1% FeCl3, 22 ml 90% lactic acid, H2O to 1 l, pH 5.5 (adjusted with KOH) and supplemented with 0.1% glucose or 0.5% galactose.
Selective media:
SD medium: 1.7 g yeast nitrogen base, 5 g (NH4)2SO4, 20 g glucose, H2O to 1 l.
SLac medium: 1.7 g yeast nitrogen base, 5 g (NH4)2SO4, 22 ml 90% lactic acid, H2O to 1 l, pH 5.5 (adjusted with KOH).
For selective media, stock solutions: histidine (10 mg/ml, 500 x stock), leucine (10 mg/ml, 333 x stock), lysine (10 mg/ml, 333 x stock), uracil (2 mg/ml, 100 x stock) and adenine (2 mg/ml, 100 x stock) were separately autoclaved for 20 min at 120°C, whereas tryptophan (10 mg/ml, 500 x stock) was filter sterilized.
The above described media were used for preparing liquid cultures. For preparation of plates with solid media, 2% w/v bacto-agar was added. Bacto-agar, glucose, and media
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were autoclaved separately. The amino acid solutions were added to the selective media just before pouring the plates.
2.2.2.2Cultivation of S.cerevisiae
Liquid cultures were inoculated with yeast strains from the glycerol stocks or from the agar plates and were grown in the appropriate liquid medium at 30°C while shaking at 140 rpm. Prior to the isolation of mitochondria cells were passaged for approximately 60 h in the way that OD578 never exceeded 1. Temperature-sensitive mutants were grown at 24°C for the same period of time. For the generation of mitochondria depleted of one its essential proteins a yeast strain having the corresponding gene under GAL promoter was grown for 48-60 h on galactose-containing media after which cells were collected, washed with water, resuspended in glucose-containing media and grown in the latter media for 8-18 h depending on the strain. For the generation of mitochondria with increased levels of one its proteins encoded on the gene under ADH promoter, the corresponding yeast strain was grown on selective lactate medium supplemented with 0.1% glucose.
2.2.2.3Transformation of S .cerivisiae by the lithium acetate method
The corresponding yeast strain was grown overnight in YPD-medium and diluted the next morning to 50 ml medium with an OD600 of 0.1-0.2. Cells were grown further, till they reached an OD600 of 0.5-0.6. Then, cells were transferred to a sterile centrifuge tube, and harvested by centrifugation (1,000×g, 3 min, RT). After washing with 25 ml of sterile water, cells were recollected, resuspended in 1 ml 100 mM lithium acetate and transferred to an Eppendorf tube. Cells were centrifuged again (7,500×g, 15 sec, RT) and were resuspended in 400 µl 100 mM lithium acetate. For each transformation 50 µl of the cell suspension was centrifuged (7,500×g, 5 min, RT) and the supernatant was removed. Next, cells were overlaid in the following order with: 240 µl PEG 3350 (50% v/v), 36 µl 1 M lithium acetate, 5 µl single stranded salmon sperm DNA (10 mg/ml; previously incubated for 5 min at 95ºC), 70 µl H2O containing 0.1-10 µg of DNA to be transformed. The mixture was vortexed for 1 min and incubated for 30 min at 30ºC, with moderate shaking, followed by another 20-25 min at 42ºC. The cells were harvested by
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centrifugation (7,000×g, 15 sec, RT), washed with sterile water, resuspended in a small volume of sterile water (150 µl), and spread on plates with the appropriate selective media. The plates were incubated for 2-4 days at 30ºC to recover transformants.
2.2.3 Isolation of mitochondria from S. cerevisiae
Mitochondria were isolated from S. cerevisiae following the previously described method (Daum et al., 1982a; Daum et al., 1982b).Yeast cells were cultivated to OD600 of 1-1.5 and collected by centrifugation (4,400×g, 5 min, RT). The pellets were washed with H2O and resuspended to a final concentration of 0.5 g/ml in DTT buffer (100 mM Tris-SO4, 10 mM dithiotreitol (DTT), pH 9.4). The cell suspension was incubated for 10 min at 30°C with gentle shaking, followed by another centrifugation step and resuspended in 100 ml of 1.2 M sorbitol buffer (1.2 M sorbitol, 20mM potassium phosphate-KOH, pH 7.4). The cell wall was digested by 2.5 mg Zymolyase per gram wet cells dissolved in Sorbitol buffer. Cells were incubated for 30-45 min at 30°C, under moderate shaking conditions. To test the cell wall digestion (obtaining of spheroplasts), 50 µl cell suspension was diluted with 2 ml H2O or into a solution of 1.2 M sorbitol. Formation of spheroplasts was complete when the OD of the H2O dilution was 10-20% of the OD of the sorbitol dilution. The solution of spheroplasts in pure H2O becomes clear because spheroplasts burst under these conditions. All the subsequent steps were performed at 4°C. The spheroplasts were isolated by centrifugation (3,000×g, 5 min, 4°C), resuspended (0.15 g/ml) in homogenization buffer (0.6 M sorbitol, 10 mM Tris-HCl, 1 mM EDTA, 0.2% (w/v) BSA, 1 mM PMSF, pH 7.4) and homogenized 10 times in a Dounce-Homogeniser. The cell remnants and unopened cells were sedimented by double centrifugation (2,000×g, 5 min, 4°C). The supernatant was spun (17,400×g, 12 min, 4°C) and the sedimented mitochondria were resuspended in SH buffer (0.6M sorbitol, 20mM HEPES, pH 7.4) and separated again from cell’s remnants (2,000×g, 5 min, 4ºC). The mitochondria were sedimented again as above (17,400×g, 12 min, 4°C). Finally, mitochondria were resuspended in a small volume of SH buffer to a concentration of 10 mg/ml protein, aliquoted, frozen in liquid nitrogen, and stored at – 80ºC till use.
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2.2.4 Isolation of crude mitochondria from S. cerevisiae
Cells corresponding to 10-20 OD units were harvested by centrifugation (800 x g, 5 min, RT) and washed with water. The cells were resuspended in 300 µl SHK buffer (0.6M sorbitol, 20mM HEPES, pH 7.4, 80mM KCl) containing 1mM PMSF and to the resuspended cells 0.3 g glass beads (diameter 0.3 mm) were added. The samples were vortexed four times 30 sec each, with 30 sec breaks in between (during this break the samples were incubated on ice) and centrifuged (1,000 x g, 3 min, 4ºC). After the centrifugation, the supernatant was transferred to a new tube and was centrifuged (10,000×g, 10 min, 4ºC), resulting in crude mitochondrial pellets.