Materials
Protocol
Results
Tips

     Common strategies for selecting transformants are based upon either prototrophic growth or drug/antibiotic resistance. In our laboratory, two different plasmid vectors have been used for evaluating transformation efficiencies. Plasmid, pAN7-1, contains the Escherichia coli hygromycin B efficiencies. Plasmid, pAN7-1, contains the Escherichia coli hygromycin B (HmB) phosphotransferase (hph) gene. Expression of the hph gene in the fungus confers resistance to antibiotic HmB. Plasmid, pWU44, contains a URA5 gene from Podospora anserina. The URA5 auxotroph of W. dermatitidis was transformed to protorophy with pWU44 by complementation. These transformation methods are being used to investigate the molecular genetics of W. dermatitidis. 

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Strains
Strain8656 (ATCC 34100; Roberts and Szaniszlo 1978), wild type. Strain Mc3W-14, a cdc mutant strain with defects also in melanin and uracil biosynthesis (cdc2, mel3-3, ura5-2; Cooper and Szaniszlo 1993; Cooper, Schafer and Szaniszlo, unpublished data).

Enzymes
Zymolyase-20T (ICN Immunobiologicals, Lisle, IL). Spheroplasting Enzyme MIXX (BIO 101, La Jolla, CA).

Antibiotics
Hygromycin B (SIGMA, St. Louis, MO)

Media
MCD: This complete medium is prepared first by adding the following components to 800 ml of distilled water: 30% (w/v) NaNo₃, 10 ml; 20% (w/v) K₂HPO₄, 5 ml; 20% (w/v) MgSO₄·7H₂0, 5 ml; 10% (w/v) KCl, 5 ml; dextrose, 30 g and adjusting to pH 6.5. Then, 1.0 g Bacto-yeast extract (Difco, Detroit, MI) and 1 ml of a freshly-prepared FeSO₄·7H₂0 (10mg·ml^-1) are added and the volume adjusted to one liter by water.

CDN: This minimal medium is prepared as MCD medium, except the yeast extract is replaced by 3 mg thiamine and 5.3g NH₅Cl for one liter of medium.

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1. Strains, 8656 and Mc3W-14, are grown for spheroplasting by inoculating yeast cells from a single colony from a maintenance culturing plate of MCD agar [MCD with 15 g Bacto agar (Difco) per liter] into 100 ml MCD and incubating with shaking at 25°C to an optimum cell density of 2 X 10⁶·ml^-1 (O.D. = 0.3). Culture is for about 3-4 days.
2. When sufficient cell mass is evident, spin to pellet cells in a 50 ml conical tube, 500 x g for 5 min, and discard supernatant
3. Resuspend in 40 ml sterile H₂0, spin to pellet cells, 500 x g for 5 min.
4. Gently resuspend cells in 40 ml Buffer A or 1 M sorbitol solution, spin 500 x g for 5 min, and discard supernatant.
5. Resuspend cells in 10 ml Buffer A and add ß-mercaptoethanol to a final concentration of 100 mM. Incubate cells for 30 min at 37°C with shaking (150 rpm) and then add 50 µl Spheroplasting Enzyme MIXX (10 mg·ml^-1), and incubate for 1 hr at 30ºC. Check spheroplasting by phase-contrast microscopy until 90-95% spheroplasting is reached.
6. Spin to pellet spheroplasts, 500 x g for 5 min and wash with 40 ml Buffer B or 1M sorbitol solution by centrifugation (500 x g for 5 min). Gently resuspend spheroplasts in 2 ml Buffer B or CaST solution and adjust to 2 X 10⁸ cells·ml^-1 final concentration of spheroplasts with the same solution. The spheroplasts are now ready for transformation.

Transformation
Plasmid pAN7-1 has been used to transform strain 8656 and plasmid pWU44 has been used to transform strain Mc3W-14, respectively

Electroporation
Electroporation experiments are conducted with Buffer B-suspended spheroplasts in 0.2 cm electrode cap cuvettes at an electrical condition of 2.6 KV·cm^-1 field strength, 200 ohms resistance, and 25 µF capacitance, corresponding to a time range of 5-10 msec, using a Gene Pulse Apparatus (Bio-Rad). Mix 5-10 µg of plasmid DNA with 400 µl of spheroplast suspension and keep on ice. Following delivery of the electrical pulse, add the suspension to 0.5 ml Buffer B, Keep on ice for 10 min and gently shake the "clump" of spheroplasts to disperse into small pieces.

PEG-mediated transformation
In a 6 ml Falcon round bottom tube (Becton Dickinson Labware, Lincoln Park, NJ), add 1 µg of carrier DNA, 5-10 µg of plasmid DNA, and 200 µl of spheroplast suspension. Gently swirl to mix. Incubate at room temperature for 20 min. Then, add 1 ml PEG solution and swirl to mix. Incubate at room temperature for 10 min. Spin, 500 x g for 5 min, discard supernatant, and resuspend in 2000 µl of 1 M sorbitol solution.

Spheroplast regeneration and transformant selection
a. After transformation, mix 200 µl of transformed spheroplast suspension with 200 µl of SOS medium and incubate at 30ºC for 40 min.

b. Mix the incubated spheroplast suspension with 5-7 ml of pre-warmed (50ºC) top agar and plate onto MCD agar containing 50-100 µg HmB·ml^-1 for selection of HmB resistant colonies transformed with pAN7-1 and CND agar for selection of prototrophs transformed with pWU44 by complementation.

DNA Isolation and Southern analysis
Putative transformant DNA is prepared as described in Ausubel et al. (1989). Digest DNA with restriction enzymes, such as EcoRI and PstI, according to the supplier’s instructions (Promaga) and electrophorese in an 0.9% agarose using a TAE buffer. Transfer this DNA to be analysed by Southern (1975) hybridization to a nytran membrane by blotting in 10 x SSPE solution and Probe the blot with a-[³²P]-labeled fragment of E. coli hph gene or with the P. anserina URA5 gene. The labeling can be accomplished usint the Primer-A –Gene System (Promaga).

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This page updated on:
Monday, March 03, 2003 11:10:07 PM