addition after the first acetone extraction, the obtained pellet
could be extracted by adding to the pellet 2.5 mL of MeOH:
CHCl 3 (1:1), and vortex until total resuspension. Centrifuge al
9000 Â g for 10 min and collect the liquid.
8. Resuspend the dry residue in CHCl 3 , CHCl 2 or acetone and
stored at À80
C prior to HPLC-PDA or HPLC-PDA-MS
analyses.
9. For volatile analyses, after induction of the cultures (point 5)
transfer 5 mL to a tightly closed 50 mL tube. Incubate the
tubes at 16
C for 20 h (125 rpm). An SPME fiber (65 μm
polydimethylsiloxane-divinylbenzene) is introduced into the
vial through a septum and the headspace volatiles are allowed
to be absorbed by the fiber at 45
C for 30 min. The SPME
fiber is keep at À80
C until the GC analyses.
3.2 In Vitro Assays
1. Amplified the CCD sequence with the corresponding specific
oligonucleotides by PCR and cloned it in the selected expression vectors.
2. Transform the competent E. coli strain selected with the expression vectors harboring the CCD under study (see Notes 3 and
4) and the empty vector that is used as a negative control of
activity (see Note 6).
3. Take individual colonies to initiate a preculture in a volume of
3 mL LB containing the appropriate antibiotics, and grow
overnight at 37
C.
4. Use 2.5 mL of the overnight cultures to inoculate 50 mL of
2ÂYT with the appropriate antibiotics at half strength. Grow
bacteria at 28
C with shaking (150 rpm) to an OD 600 nm of
0.7, and induced with the appropriate agent (arabinose or
IPTG), depending on the expression vector selected
(Table 3). Incubate the cultures at 28
C with shaking
(150 rpm) for 4-6 h or overnight at 18–20
C (see Note 5).
5. Harvest the E. coli cells by centrifugation for 6 min at 6000 Â g.
Resuspend the cell pellet in 1 mL 2Â LEW buffer, and incubate
the tubes for 30 min on ice. Disrupt the cells by sonication,
French pressing, or other preferred method [55], and centrifuged at 12,000 Â g for 30 min at 4
C. Use the isolated
supernatant for the in vitro assays.
6. Prepare the carotenoid and or apocarotenoid substrates for the
assays. See item 6 in Subheading 2.2.
7. For the in vitro assay. In a total volume of 200 μL, the carotenoid gel is resuspended by vortex in 100 μL 2Â assay incubation
buffer (single assay) and sonicated to obtain a clear micellar
solution. Add 50 μL of crude protein lysate and ddH 2 O to
adjust at 200 μL. Transfer the tubes into a shaker (200 rpm) at
Determination of In Vitro and In Vivo Activities of Plant Carotenoid. . .
69
could be extracted by adding to the pellet 2.5 mL of MeOH:
CHCl 3 (1:1), and vortex until total resuspension. Centrifuge al
9000 Â g for 10 min and collect the liquid.
8. Resuspend the dry residue in CHCl 3 , CHCl 2 or acetone and
stored at À80
C prior to HPLC-PDA or HPLC-PDA-MS
analyses.
9. For volatile analyses, after induction of the cultures (point 5)
transfer 5 mL to a tightly closed 50 mL tube. Incubate the
tubes at 16
C for 20 h (125 rpm). An SPME fiber (65 μm
polydimethylsiloxane-divinylbenzene) is introduced into the
vial through a septum and the headspace volatiles are allowed
to be absorbed by the fiber at 45
C for 30 min. The SPME
fiber is keep at À80
C until the GC analyses.
3.2 In Vitro Assays
1. Amplified the CCD sequence with the corresponding specific
oligonucleotides by PCR and cloned it in the selected expression vectors.
2. Transform the competent E. coli strain selected with the expression vectors harboring the CCD under study (see Notes 3 and
4) and the empty vector that is used as a negative control of
activity (see Note 6).
3. Take individual colonies to initiate a preculture in a volume of
3 mL LB containing the appropriate antibiotics, and grow
overnight at 37
C.
4. Use 2.5 mL of the overnight cultures to inoculate 50 mL of
2ÂYT with the appropriate antibiotics at half strength. Grow
bacteria at 28
C with shaking (150 rpm) to an OD 600 nm of
0.7, and induced with the appropriate agent (arabinose or
IPTG), depending on the expression vector selected
(Table 3). Incubate the cultures at 28
C with shaking
(150 rpm) for 4-6 h or overnight at 18–20
C (see Note 5).
5. Harvest the E. coli cells by centrifugation for 6 min at 6000 Â g.
Resuspend the cell pellet in 1 mL 2Â LEW buffer, and incubate
the tubes for 30 min on ice. Disrupt the cells by sonication,
French pressing, or other preferred method [55], and centrifuged at 12,000 Â g for 30 min at 4
C. Use the isolated
supernatant for the in vitro assays.
6. Prepare the carotenoid and or apocarotenoid substrates for the
assays. See item 6 in Subheading 2.2.
7. For the in vitro assay. In a total volume of 200 μL, the carotenoid gel is resuspended by vortex in 100 μL 2Â assay incubation
buffer (single assay) and sonicated to obtain a clear micellar
solution. Add 50 μL of crude protein lysate and ddH 2 O to
adjust at 200 μL. Transfer the tubes into a shaker (200 rpm) at
Determination of In Vitro and In Vivo Activities of Plant Carotenoid. . .
69