Transformation of Aspergillus fumigatus by electroporation

Introduction

This procedure allows transformation of A. fumigatus with efficiencies averaging 200 transformants/µg DNA. Efficiencies up to 1000 transformants/µg have been observed. It is adapted from the protocol of O. Sanchez and J. Aguirre developed for A. nidulans.

Materials

• YG medium : 0.5% Yeast Extract, 2% D-glucose supplemented with 5 mM uridine and 5mM uracil when a pyrG strain is used
• ice-cold sterile water
• YED pH 8.0 : 1% yeast extract, 1% glucose, 20 mM HEPES, adjust pH to 8.0 with 1 M NaOH)
• EB (ice-cold) : 10 mM Tris-HCl pH 7.5, 270 mM sucrose, 1 mM Li-acetate
• Selective medium : Aspergillus minimal medium (0.52 g/l KCl, 0.52 g/l MgSO₄.7H₂O, 1,52 g/l KH₂PO₄, trace elements 1X, 1% D-glucose, 5 mM Na-glutamate, adjust to pH 6.8 with 1 M NaOH, 1.5% Oxoid agar) when a pyrG strain is used

Equipment

• Rotary shaker at 37°C
• Rotary shaker at 30°C
• Table-top centrifuge
• BioRad electroporation device (settings 1 kV, 400 W, 25µF)
• 0.2 cm BioRad electroporation cuvettes
• 37°C incubator

Procedure

1. Inoculate 125 ml YG medium at 10⁷ spores/ml with washed conidia. Conidia are collected from A. fumigatus lawns grown on complete medium agar plates or slants (3-4 days at 37°C) and resuspended in PBS.Tween 20 0.1%, filtered and washed 5 times in sterile distilled water.
2. Grow for 4 hr at 37°C at 300 rpm on rotary shaker
3. Collect swollen spores by centrifugation 5 min 4000 x g at 4°C
4. Resuspend spores in 200 ml ice-cold sterile water, centrifuge 5 min at 4000 x g at 4°C
5. Resuspend spores in 12.5 ml YED pH 8.0 and incubate 60 min at 30C at 100 rpm on rotary shaker
6. Centrifuge 5 min at 4000 x g
7. Resuspend spores in 1 ml ice-cold EB at 10⁹ conidia/ml and keep on ice
8. Mix 50 µl conidial suspension with 1 to 2 µg DNA in a total volume of 60µl in Epp. tubes and keep on ice for 15 min
9. Transfer suspension to 0.2 cm electroporation cuvette and electroporate with BioRad instrument
10. Add 1 ml ice-cold YED and transfer to pre-cooled sterile 15 ml tube, keep on ice for 15 min
11. Incubate at 30°C for 90 min at 100 rpm on rotary shaker, tubes in horizontal position
12. Spread 100, 200, and 700 µl on minimal medium agar plates and incubate at 37°C
13. Transformants are observed after 36-48 hours at 37°C

Timetable

Tips and general comments

1. Freshly harvested conidia should be used to ensure highest transformation efficiencies.
2. As is usual with transformation of filamentous fungi, highest transformation efficiencies are obtained with a linearized vector. Targeted integration appears to occur at a lower frequency than that observed following protoplast transformation when this protocol is used.
3. This protocol has been used with different markers including pyrG and hyg^R. With the latter, selection of transformants can be obtained by plating the transformation mixture directly on hygromycin-containing plates or by plating the cells on non-selective medium, incubating ON at 37°C and then overlaying the plates with hygromycin-containing soft agar (0.7%) medium. The final hygromycin concentration in the plate should be 200 µg/ml.
4. A pyrG strain can be obtained upon written request from Christophe d'Enfert, Laboratoire des Aspergillus, Institut Pasteur, 75724 Paris Cedex 15, France.

References

Sanchez, O. and J. Aguirre. (1996). Efficient transformation of Aspergillus nidulans by electroporation of germinated conidia. Fungal Genetics Newsletter 43: 48-51.

Weidner, G., d'Enfert, C., Koch, A., Mol, P., and Brakhage, A.A. (1998) Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine monophosphate decarboxylase. Current Genet. 33: 378-385.

back to index