GGGCAAGCATTGTCA-3
0 . PmeI restriction sites are in
italics.
5. Escherichia coli competent cells (DH5α): Φ-80
0
ΔlacZM15
Δ(lacZYA-argF)U169, gyrA96 recA1 endA1 thi-1, hsdR17
(rkÀ, mk+), supE44, rel A1, deo R.
6. LB medium: 5 g yeast extract, 10 g Bacto tryptone, 10 g NaCl
in 1 l of distilled water.
7. LB-Amp agar medium: 5 g yeast extract, 10 g Bacto tryptone,
10 g NaCl, and 16 g agar in 1 l of distilled water. Autoclave.
Cool until 55
C. Add 100 μl of 100 mg/ml sodium ampicillin.
8. LB-IPTG-X-Gal-Amp medium: Prepare LB-Amp agar and
supplement with 0.5 mM IPTG and 85 μg/ml X-Gal.
9. T4 DNA ligase.
10. Expand High Fidelity PCR System (Roche).
11. Restriction enzymes PmeI, SacII, and EcoRI.
12. DNA size marker.
13. TAE 50Â buffer: 2 M Tris–acetate, pH 8.0, EDTA 0.05 M.
14. Isolate II PCR and Gel Kit (Bioline).
15. NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).
16. Horizontal electrophoresis system.
17. Power supply.
2.2 Cultivation of
Fusarium fujikuroi
Sterilize all media and material in an autoclave at 120
C for 20 min.
1. F. fujikuroi IMI 58289 wild type strain (previously, Gibberella
fujikuroi), obtained from the Imperial Mycological Institute,
Kew, UK.
2. F. fujikuroi strain SG39. A superproducer of carotenoids with a
mutation in the gene carS [19].
3. DG medium [20]: 30 g of glucose, 3 g of NaNO 3 , 1 g of
H 2 KPO 4 , 0.5 g of MgSO 4 ·7H 2 O, 0.5 g of KCl, 16 g of agar,
and 2 ml of microelement solution in 1 l of distilled water.
Autoclave. This is the minimal medium for F. fujikuroi.
4. Microelement solution for DG medium: 0.5 mg of HBO 4 ,
5 mg of CuSO 4 , 10 mg of FeCl 3 , 1 mg of MnCl 2 , 1 mg of
NaMoO 4 , and 100 mg of ZnSO 4 in 100 ml of distilled water.
5. Medium EG [21]: 1 g glucose, 1 g yeast extract, 1 g NH 4 NO 3 ,
1 g KH 2 PO 4 , 0.5 g SO 4 Mg·7H 2 O, 16 g agar in 1 l of distilled
water. Autoclave. Medium EG was used to obtain spores.
6. Sterile VitraPOR
® borosilicate filter crucibles, porosity
1, 15-ml volume, ROBU
® (Glasfilter-Ger€ ate GmbH, Hattert,
Germany).
346
Julia Marente et al.
0 . PmeI restriction sites are in
italics.
5. Escherichia coli competent cells (DH5α): Φ-80
0
ΔlacZM15
Δ(lacZYA-argF)U169, gyrA96 recA1 endA1 thi-1, hsdR17
(rkÀ, mk+), supE44, rel A1, deo R.
6. LB medium: 5 g yeast extract, 10 g Bacto tryptone, 10 g NaCl
in 1 l of distilled water.
7. LB-Amp agar medium: 5 g yeast extract, 10 g Bacto tryptone,
10 g NaCl, and 16 g agar in 1 l of distilled water. Autoclave.
Cool until 55
C. Add 100 μl of 100 mg/ml sodium ampicillin.
8. LB-IPTG-X-Gal-Amp medium: Prepare LB-Amp agar and
supplement with 0.5 mM IPTG and 85 μg/ml X-Gal.
9. T4 DNA ligase.
10. Expand High Fidelity PCR System (Roche).
11. Restriction enzymes PmeI, SacII, and EcoRI.
12. DNA size marker.
13. TAE 50Â buffer: 2 M Tris–acetate, pH 8.0, EDTA 0.05 M.
14. Isolate II PCR and Gel Kit (Bioline).
15. NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).
16. Horizontal electrophoresis system.
17. Power supply.
2.2 Cultivation of
Fusarium fujikuroi
Sterilize all media and material in an autoclave at 120
C for 20 min.
1. F. fujikuroi IMI 58289 wild type strain (previously, Gibberella
fujikuroi), obtained from the Imperial Mycological Institute,
Kew, UK.
2. F. fujikuroi strain SG39. A superproducer of carotenoids with a
mutation in the gene carS [19].
3. DG medium [20]: 30 g of glucose, 3 g of NaNO 3 , 1 g of
H 2 KPO 4 , 0.5 g of MgSO 4 ·7H 2 O, 0.5 g of KCl, 16 g of agar,
and 2 ml of microelement solution in 1 l of distilled water.
Autoclave. This is the minimal medium for F. fujikuroi.
4. Microelement solution for DG medium: 0.5 mg of HBO 4 ,
5 mg of CuSO 4 , 10 mg of FeCl 3 , 1 mg of MnCl 2 , 1 mg of
NaMoO 4 , and 100 mg of ZnSO 4 in 100 ml of distilled water.
5. Medium EG [21]: 1 g glucose, 1 g yeast extract, 1 g NH 4 NO 3 ,
1 g KH 2 PO 4 , 0.5 g SO 4 Mg·7H 2 O, 16 g agar in 1 l of distilled
water. Autoclave. Medium EG was used to obtain spores.
6. Sterile VitraPOR
® borosilicate filter crucibles, porosity
1, 15-ml volume, ROBU
® (Glasfilter-Ger€ ate GmbH, Hattert,
Germany).
346
Julia Marente et al.