2. Check periodically the formation of protoplasts during the
incubation of mycelia with enzymes by observing 10-μl aliquots under microscope (200Â). Protoplasts, visible as round
cells, should start appearing after 30–60 min of incubation.
3. Never use pipette tips to resuspend protoplast pellets. Instead,
disaggregate gently the pellet with smooth taps. From this
moment on, use cut pipette tips along all the protocol to
avoid burst of protoplasts.
4. Competence of protoplasts tends to decrease upon storing at
À80
C.
5. In our case, simultaneous transformation with two plasmids
(cotransformation) was done because the selection marker was
not included in the plasmid containing the Tet-on cassette.
6. Prepare 1Â regeneration medium by mixing 60 ml of warm
2Âregeneration medium with 60 ml of warm 1.6 M sucrose.
Keep the medium in a bath at 55
C.
7. Spores (microconidia) are uninucleate. Single-spore purification allows obtaining pure clones in the case of heterokaryotic
or unstable transformants.
8. F. fujikuroi IMI58289 sporulates poorly. Inoculate at least
three Petri dishes for each strain to ensure collecting enough
spores.
9. Because of conidia hydrophobicity, use of glycerol is recommended to facilitate suspension of the conidia.
10. If borosilicate filters are not available, the conidia suspension
may be passed through a standard Whatman filter paper.
11. The optimal concentration of doxycycline for our growth conditions was formerly determined mixing 50 μl with 10
4 fresh
spores of the wild type IMI58289 or the carS mutant SG39
and 50 μl of 2Â DGpeptone in a 96-well plate. Doxycycline
was added at different concentrations (from 10 to 160 μg/ml)
after 15 h incubation at 30
C and growth was checked after
72 h (without shaking). Optimal time for doxycycline additions was also formerly determined for the expression analysis
experiments.
12. Wear gloves during RNA extraction and subsequent manipulations to avoid degradation.
13. Calculate the volume of the RT mix considering three replicates for each of the investigated genes (the genes of interest
and the reference gene(s)). Add some additional volume to
prevent pipetting error.
14. Gene for β-tubulin was used as a reference.
Modulated Expression of a Key Regulatory Gene in Carotenoid Biosynthesis
357
incubation of mycelia with enzymes by observing 10-μl aliquots under microscope (200Â). Protoplasts, visible as round
cells, should start appearing after 30–60 min of incubation.
3. Never use pipette tips to resuspend protoplast pellets. Instead,
disaggregate gently the pellet with smooth taps. From this
moment on, use cut pipette tips along all the protocol to
avoid burst of protoplasts.
4. Competence of protoplasts tends to decrease upon storing at
À80
C.
5. In our case, simultaneous transformation with two plasmids
(cotransformation) was done because the selection marker was
not included in the plasmid containing the Tet-on cassette.
6. Prepare 1Â regeneration medium by mixing 60 ml of warm
2Âregeneration medium with 60 ml of warm 1.6 M sucrose.
Keep the medium in a bath at 55
C.
7. Spores (microconidia) are uninucleate. Single-spore purification allows obtaining pure clones in the case of heterokaryotic
or unstable transformants.
8. F. fujikuroi IMI58289 sporulates poorly. Inoculate at least
three Petri dishes for each strain to ensure collecting enough
spores.
9. Because of conidia hydrophobicity, use of glycerol is recommended to facilitate suspension of the conidia.
10. If borosilicate filters are not available, the conidia suspension
may be passed through a standard Whatman filter paper.
11. The optimal concentration of doxycycline for our growth conditions was formerly determined mixing 50 μl with 10
4 fresh
spores of the wild type IMI58289 or the carS mutant SG39
and 50 μl of 2Â DGpeptone in a 96-well plate. Doxycycline
was added at different concentrations (from 10 to 160 μg/ml)
after 15 h incubation at 30
C and growth was checked after
72 h (without shaking). Optimal time for doxycycline additions was also formerly determined for the expression analysis
experiments.
12. Wear gloves during RNA extraction and subsequent manipulations to avoid degradation.
13. Calculate the volume of the RT mix considering three replicates for each of the investigated genes (the genes of interest
and the reference gene(s)). Add some additional volume to
prevent pipetting error.
14. Gene for β-tubulin was used as a reference.
Modulated Expression of a Key Regulatory Gene in Carotenoid Biosynthesis
357