7. 2Â Modified LEW (Lysis-Equilibration-Wash) buffer:
100 mM NaH 2 PO 4 , 600 mM NaCl, 1 mg/mL lysozyme,
0.1% Triton X-100 and 1 mM DTT. Adjust pH to 8.0
with NaOH.
8. Detergents:
Triton
X-100,
Triton
X-405,
and
octyl-β-glucoside.
9. Organic solvents for extraction and substrates preparation:
acetone, benzene, CHCl 2 , CHCl 3 , petroleum, ether petroleum, and diethyl ether.
10. Expression vectors. Most frequently used expression vectors
are present in Table 3.
11. 20% Arabinose dissolved in ddH 2 O and sterilized by filtration.
12. 1 mM isopropyl β-d-thiogalactopyranoside (IPTG) dissolved
in ddH 2 O and sterilized by filtration.
13. 2Â Assay incubation buffer: 200 mM HEPES–NaOH, pH 7.8,
2 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP),
0.4 mM FeSO 4 , 2 mg mL/L catalase.
14. Vials and columns for HPLC and GC analyses.
2.3 Equipment
1. Table centrifuge.
2. Shaker with controlled temperature.
3. Spectrophotometer.
4. French pressure cell or an ultrasonic homogenizer.
5. Vacuum centrifuge.
6. Nitrogen.
7. Balance.
8. HPLC-PDA.
Table 3
Expression vectors and conditions used for in vitro characterization of CCDs activities
Expression vector
Resistance
Induction
References
pGEX-2T
Ampicillin
0.2 mM IPTG
[43]
pGEX-4T1
Ampicillin
0.2 mM IPTG
[42]
pGEX-4T1
Ampicillin
No induction
[47, 51]
pThio-DAN2
Ampicillin
0.2% arabinose
[22, 24–26, 52]
pBAD/THIO-TOPO
® TA
Ampicillin
0.2% arabinose
[46]
pGEX 5X-2
Ampicillin
0.2 mM IPTG
[35, 36]
pGEX 5X-3
Ampicillin
0.2 mM IPTG
[44]
pGEX-6P-1
Ampicillin
0.1 mM IPTG
[41]
Determination of In Vitro and In Vivo Activities of Plant Carotenoid. . .
67
100 mM NaH 2 PO 4 , 600 mM NaCl, 1 mg/mL lysozyme,
0.1% Triton X-100 and 1 mM DTT. Adjust pH to 8.0
with NaOH.
8. Detergents:
Triton
X-100,
Triton
X-405,
and
octyl-β-glucoside.
9. Organic solvents for extraction and substrates preparation:
acetone, benzene, CHCl 2 , CHCl 3 , petroleum, ether petroleum, and diethyl ether.
10. Expression vectors. Most frequently used expression vectors
are present in Table 3.
11. 20% Arabinose dissolved in ddH 2 O and sterilized by filtration.
12. 1 mM isopropyl β-d-thiogalactopyranoside (IPTG) dissolved
in ddH 2 O and sterilized by filtration.
13. 2Â Assay incubation buffer: 200 mM HEPES–NaOH, pH 7.8,
2 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP),
0.4 mM FeSO 4 , 2 mg mL/L catalase.
14. Vials and columns for HPLC and GC analyses.
2.3 Equipment
1. Table centrifuge.
2. Shaker with controlled temperature.
3. Spectrophotometer.
4. French pressure cell or an ultrasonic homogenizer.
5. Vacuum centrifuge.
6. Nitrogen.
7. Balance.
8. HPLC-PDA.
Table 3
Expression vectors and conditions used for in vitro characterization of CCDs activities
Expression vector
Resistance
Induction
References
pGEX-2T
Ampicillin
0.2 mM IPTG
[43]
pGEX-4T1
Ampicillin
0.2 mM IPTG
[42]
pGEX-4T1
Ampicillin
No induction
[47, 51]
pThio-DAN2
Ampicillin
0.2% arabinose
[22, 24–26, 52]
pBAD/THIO-TOPO
® TA
Ampicillin
0.2% arabinose
[46]
pGEX 5X-2
Ampicillin
0.2 mM IPTG
[35, 36]
pGEX 5X-3
Ampicillin
0.2 mM IPTG
[44]
pGEX-6P-1
Ampicillin
0.1 mM IPTG
[41]
Determination of In Vitro and In Vivo Activities of Plant Carotenoid. . .
67