Team:NYMU-Taipei/Notebook-Lab Book-prototype lab book
Glass jar containing Metarhizium anisopliae ARSEF 549 was unsealed. Sterile ddH2O was added to revitalize freeze dried fungi. A part of the sample were streaked onto PDA plates and incubated in incubator at 25°C. The rest of the sample was placed in -80°C refrigerator for storage.
Conidia were collected for one M. anisopliae plate from 07/15 and cultured in PD broth.
Created first generation fungal cells of M. anisopliae glycerol-frozen sample—
Freeze protectant formula: 20%(v/v) glycerol, 10%(w/v) lactose solution, 70%(v/v) ddH2O
Every cryovial contains 2mL freeze protectant.
Fungal sample freeze method:
1. Use hole puncher to punch holes at the rim of the colony
2. Collect samples with platinum needles and place in the cryovial
3. Place cryovial containing fungal samples into -80°C refrigerator for storage
Two 100mL PDB liquid cultures in cones bottles of M. anisopliae were prepared. The cultures were incubated at 26°C, 150rpm for two days.
Begin tests for effective fungal selection marker (Candidate antibiotics: hygromycin and phleomycin)
Test plates (antibiotic concentration):
1. Hygromycin (μg/mL): 0, 50, 100, 150, 200
2. Phleomycin (μg/mL): 0, 25, 50
Antibiotic stock solution formula:
1. Hygromycin (100 mg/mL): 250mg hygromycin powder + 2.5mL sterile ddH2O
2. Phleomycin (100 mg/mL): 250mg phleomycin powder + 2.5mL sterile ddH2O
M. anisopliae were incubated in each of the antibiotics test plates and incubated at 25°C.
Performed DNA extraction on the two-day-old fungal culture plated on 8/15 to extract M. anisopliae genomic DNA.
M. anisopliae genomic DNA extraction;
Two cones bottles of 100mL PDB liquid culture for protoplast production were prepared. The cultures were grown overnight (12-16hr).
Make M.anisopliae protoplast for protoplast transformation;
Prepared 2 cones bottles of 100mL of M. anisopliae PDB liquid culture for the genomic DNA extraction on 9/2;
Produced M. anisopliae protoplasts for protoplast transformation.
Performed protoplast transformation,we transformed the plasmid pBGRFP into our fungi;
Performed protoplast transformation,we transformed the plasmid pBGRFP into our fungi;
Performed protoplast transformation;
Produced protoplasts with the concentrations of: 3 X 106 protoplasts/mL, 3.6 X 106 protoplasts /mL. Protoplasts were stored in -80°C refrigerator.
M. anisopliae conidia were collected for Bactrocera dorsalis infection test. 3 tubes of conidia suspension solution (Concentrations: 1.6 X 104 conidia/mL, 1.6 X 105 conidia/mL, 1.6 X 106 conidia/mL) were created by calculating the concentration of initial conidia suspension with light microscopes and cytometers, then dilute each solution to adjust the concentration.
Transformed the linear vector pBAR_KR into M.anisopliae protoplasts.
Protein extraction from Killer Red transformed E.coli and wild type E.coli.
Thermal stability assay of Killer Red.
