Microbiological methods

2.10.1 Heat shock transformation of Escherichia coli

A single fresh colony of E. coli DH5α cells was inoculated into 50 ml LB medium and grown over night at 37 °C with shaking (220 rpm). Five ml of the starter culture was used to inoculate 500 ml LB medium, which was then grown at 37 °C, 220 rpm to an OD₆₀₀ of ~0.4.

After cooling on ice the cells were harvested by centrifugation (15 min, 4 °C, 2500 x g), the pellet was resuspended in 250 ml ice cold 100 mM CaCl₂, centrifuged again and then resuspended in 50 ml 100 mM CaCl₂. After another centrifugation, cells were resuspended in 5 ml 100 mM CaCl₂, 20% (w/v) glycerol and 100 µl aliquots were transferred into prechilled, sterile polypropylene tubes. Cells were frozen immediately in liquid nitrogen and stored at -70

°C until use. One aliquot (100 µl) of chemically competent DH5α-cells was thawed on ice and gently mixed with 5-15 ng of DNA in prechilled polypropylene tubes. After incubation on ice for 10 min, cells were incubated at 42 °C for 1 min, resuspended in 900 µl dYT-medium and incubated at 37 °C for 45 min to recover. Afterwards cells were plated on selective media (LB + antibiotics) and incubated at 37 °C over night. Transformants were picked the next day.

2.10.2 Electrotransformation of Escherichia coli

Electrotransformation was used to introduce large plasmids (e.g. pSFS1A complementation constructs) into E.coli. 500ml dYT medium were inoculated with 5 ml of an overnight culture of TOP10 cells and grown until mid-log phase (OD₆₀₀ ~0.6) at 37°C, 220 rpm. The culture was cooled on ice for about 30 min before the cells were pelleted in the centrifuge (20 min, 4000xg, 4°C). The pellet was washed once with 200 ml ice-cold sterile ddH₂O and once with 40 ml ice-cold 10% (v/v) glycerol. Cells were finally resuspended in 2 ml ice-cold 10% (v/v) glycerol and 50 μl aliquots were frozen in liquid nitrogen and stored at -80°C until use. For electroporation cells were thawed on ice before they were gently mixed with the purified plasmid DNA and transferred into a prechilled electroporation cuvette (2 mm). The electroporation was performed at 1.6 kV, 186 Ω, 25 µF. Immediately after the pulse 950 µl

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SOC medium was added and bacteria were recovered at 37 °C for 30 min before plating on selective media.

2.10.3 Escherichia coli colony PCR

To screen transformants for successful insert integration a colony PCR was performed using material of a freshly grown colony directly in the PCR mixture. PCR was performed as described in 3.8.1.

2.10.4 Heat shock transformation of Candida glabrata

We used a transformation protocol based on a method that was described previously (WALTHER and WENDLAND, 2003). In brief: an overnight culture was used to inoculate 50 ml YPD at an OD₆₀₀ of 0.2. Cells were grown at 37 °C, 220 rpm until they reached an OD₆₀₀ of 1.0. Cells were pelleted (4000 x g, 4 °C) and were washed twice with 30 ml ice cold, sterile water and once with 30 ml ice cold TEL (10 mM Tris, 1 mM EDTA, 100 mM lithium acetate, pH 7.5). The cell pellet was resuspended in 1.5 ml TEL and used for transformation. The transformation mixture contained 100 µl cells, 100 µg (10 µl) Yeast Maker Carrier DNA (Clontech), 600 µl 40% (w/v) Polyethylene glycol 4000 in TEL and 2-5 µg DNA. After mixing, the transformation mixture was incubated overnight at 30 °C. Heat shock was performed at 44 °C for 15 min. Cells were pelleted at 800 x g and were subsequently resuspended in 200 µl TE (10 mM Tris, 1 mM EDTA, pH 7.5). Cells were plated on selective plates and were incubated at 37 °C. Transformants could be picked after one to two days.

2.10.5 Electrotransformation of Candida glabrata Solutions used for the electrotransformation :

10 x TE 10 x LiAc

100 mM Tris-HCl pH 8 1 M lithiumacetate pH 7.5 10 mM EDTA

To improve transformation efficiency for larger DNA fragments, electro transformation was performed as an alternative. C. glabrata cells were grown in 200 ml YPD at 37 °C, 220 rpm to mid log phase (OD₆₀₀ ~0.6). The cells were pelleted at 5000 x g and resuspended in 40 ml water before 5 ml 10 x TE and 5 ml 10 x LiAc were added. The suspension was incubated for 45 min at 30 °C, shaking with 150 rpm prior to the addition of 1.25 ml 1 M DTT. The incubation was continued for another 15 min. Afterwards, 200 ml water was added to wash

MATERIALS AND METHODS 53 the cells. Following the washing, cells were sedimented at 5000 x g and washed with 125 ml of ice cold water. Cells were then washed with 20 ml of ice cold 1 M sorbitol. After pelleting at 5000 x g, the cells were resuspended in 250 µl of 1 M sorbitol. Freshly prepared competent cells were used for transformation by electroporation.

For the transformation of C. glabrata with constructs for gene knock out or complementation the corresponding cassettes were mobilized from the plasmid with restriction enzymes. The DNA was purified and used for transformation. In prechilled polypropylene tubes 40 µl of freshly prepared competent cells were mixed with 3 to 5 µg DNA. This mixture was transferred to prechilled 2 mm electroporation cuvettes and incubated on ice for 30 min.

Transformation was performed in a BTX electroporation system at 186 Ω, 1.6 kV and 25 µF.

After the pulse cuvettes were immediately rinsed with 950 ml YPD and cells were incubated for 2 hours at 37 °C, 220 rpm to recover before they were plated on selective plates, which were either YNB drop out plates for (non-) auxotrophic mutants or YPD plates with nurseothricin for transformations with the flipper-construct. Plates were incubated at 37 °C for 1-3 days until colonies appeared.

2.10.6 Yeast colony PCR

To screen for positive transformants a colony PCR was performed with a crude cell extract of the transformants. For this, a medium sized colony was taken off the plate with a pipette tip and the tip was rinsed in 20 µl zymolyase solution (2.5 mg/ml zymolyase in 1.2 M sorbitol, 0.1 M Na₃PO₄). The suspension was incubated at 37 °C for 30 min and boiled at 95 °C for 10 min. The prepared cell extract was chilled on ice, before 2 µl were used in the PCR reaction.

2.10.7 Culturing of Candida glabrata for proteomic analysis

Single colonies of C. glabrata ATCC2001 were inoculated into YPD medium and grown for 24 h at 37 °C. Four single clonal cultures were pooled, washed and resuspended in sterile water and used to inoculate 600 ml of Pan Fungal Minimal Media (PFM) pH 7.4 at a density of 8 x 10⁵ cfu/ml. C. glabrata PFM cultures were grown at 37 °C, 220 rpm for 16-18 hours until they reached an OD₆₀₀ of 0.5. Cells were collected by centrifugation (5 min, 800 x g) and shifted to PFM at pH 4.0, 7.4 or 8.0 at the same optical density. Cultures were grown at 37 °C, 220 rpm for 2 hours and harvested by centrifugation (5 min, 800 x g, 4 °C). Pellets were washed twice with ice cold water and stored at -80 °C until used for protein extraction.

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