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Team:Goettingen/protocol inVivo
From 2014.igem.org
Overview
PCR Methods
Plasmid Construction
- Restriction of DNA
- Ligation of DNA fragments
- BP recombination reaction
- LR recombination reaction
- SEAMLESS Cloning
- Peptide Library construction
Plasmid Transformation
- E.coli competent cells
- Plasmid isolation (E.coli)
- E.coil transformation
- Plasmid isolation (Yeast)
- Yeast transformation
Colony Scanning
Protein Assessment
In vivo tests
General information:
| Strains for experiments: | Used peptide: |
|---|---|
| Aspergillus fumigatus: AfS35 (wild type) | 13 |
| Aspergillus nidulans: A4 (wild type) | 13 |
| Candida glabrata: ATCC2001 (wild type) | 4, 5 |
| Candida glabrata Δssr1: ATCC2001 (CAGL0H06413g) | 4 |
Aspergillus nidulans and Aspergillus fumigatus were washed with 0.96% NaCl and grown in minimal media at 37°C.
Candida glabrata was washed with PBS and grown in YPAD at 30°C.
Production of an A. fumigatus and A. nidulans spore suspension:
1. Plate 10 µl of an A. fumigatus respectively A. nidulans spore suspension were on minimal media plates.
2. Incubate the plates for 3 days at 37 °C.
3. Afterwards, wash off the spores with 5 ml NaCl-Tween (0.96% NaCl, 0.02% Tween80).
Cultivation of Aspergillus for in vivo experiments:
1. Inoculate 50 ml minimal medium in baffeld flasks with 20 µl spores of Aspergillus fumigatus or Aspergillus nidulans..
2. Incubate the cell suspensions at 37 °C over night on a shaker at least 160 rpm.
Cultivation of Candida glabrata for in vivo experiments:
1. Inoculate 50 ml YPAD medium with desired strains.
2. Incubate the cells at 30 °C over night on a shaker.
Immunofluorescence Staining
1. Use a stain expressing the protein of interest and a mutant strain without the protein as negative control (if available) and cultivate them over night (see cultivation of Aspergillus).
2. Centrifuge of 1 ml of each over night culture.
3. Wash the cells 3 times with NaCl or PBS and resuspend them in 500 µl minimal medium or YPAD.
2. Add 20 µl peptide solution (2-3 mg/ml) to the samples and incubate for at least 1 hour 37 °C or at 30°C on a shaker.
3. Wash the cells again 3 times with 500 µl NaCl or PBS, centrifuge them at 8000 rpm for 2 minutes and resuspend in 500 µl minimal media or YPAD.
3. Add 1 µl StrepMAB Classic Chromeo 488 antibody to the samples and incubated for 1 hour at room temperature in the dark.
5. Wash 3 times with 500 µl PBS and resuspend in minimal medium or PBS and take pictures with the fluorescence microscope.
Calcofluor staining
1. Add 1 µl Calcofluor stock solution (10mg/ml) to 1 ml cell suspension (after immunofluorescence staining) and incubate for 2 minutes at room temperature.
2. Aspirate the staining solution and wash the cells three times in 1 ml PBS. Then resuspend the cells in 500 µl minimal media or YPAD.
3. Analyze the cells with a DAPI filter (365-395 nm) under the fluorescence microscope.
