until the solutes have dissolved. Adjust the pH to 7.0 with 5 N
NaOH. Adjust the final volume of the solution to 1 L with
ddH 2 O. Sterilize by autoclaving for 20 min at 15 psi. For solid
LB medium, add 15 g of agar.
4. Antibiotics stocks. Ampicillin, carbenicillin, and kanamycin are
dissolved in ddH 2 O at a concentration of 100 mg/mL for
ampicillin and 50 mg/mL for carbenicillin and kanamycin.
Chloramphenicol is dissolved in EtOH at a concentration of
50 mg/mL.
5. Organic solvents for carotenoid and apocarotenoid extraction
and analysis. Acetone, MeOH, CHCl 2 , CHCl 3 .
6. Specific oligonucleotides for cloning in an expression vector
the CCD to be characterized.
7. Expression vectors. Most frequently used expression vectors
are shown in Table 2.
8. 20% arabinose dissolved in ddH 2 O and sterilized by filtration.
9. 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) dissolved
in ddH 2 O and sterilized by filtration.
10. Nitrogen.
11. Vials and columns for HPLC and GC analyses.
Table 1
Plasmids for carotenoids production in E. coli
Plasmid name Genes present
Carotenoid
produced
pAC-PHYTipi Contains crtE, crtB, and idi genes of Erwinia herbicola (Pantoea
agglomerans) Eho10
Phytoene
pAC-ZETAipi Contains crtE, crtB, and idi genes of Erwinia herbicola (Pantoea
agglomerans) Eho10, and crtP gene of Synechococcus PCC7942
ζ-Carotene
pAC-NEUR
Contains the Erwinia herbicola (Pantoea agglomerans) Eho10 genes
crtE and crtB, and the Rhodobacter capsulatus crtI gene
Neurosporene
pAC-LYCipi
Contains crtE, crtB, crtI, and idi genes of Erwinia herbicola (Pantoea
agglomerans) Eho10
Lycopene
pAC-DELTA
Contains crtE, crtB, and crtI genes of Erwinia herbicola (Pantoea
agglomerans) Eho10, together with the lcyE gene of Arabidopsis
thaliana
δ-Carotene
pAC-BETAipi Contains ctrE, crtB, crtI, crtY, and idi genes of Erwinia herbicola
(Pantoea agglomerans) Eho10
β-Carotene
pAC-ZEAXipi Contains crtE, idi, crtI, crtY, crtB, and crtZ genes of Erwinia herbicola
(Pantoea agglomerans) Eho10
Zeaxanthin
pAC-VIOL
Contains crtE, idi, crtI, crtY, crtB, and crtZ genes of Erwinia herbicola
(Pantoea agglomerans) Eho10 and ZEP from Arabidopsis thaliana
Violaxanthin
All the plasmids are low copy bacterial cloning vectors and provide chloramphenicol resistance
Determination of In Vitro and In Vivo Activities of Plant Carotenoid. . .
65
NaOH. Adjust the final volume of the solution to 1 L with
ddH 2 O. Sterilize by autoclaving for 20 min at 15 psi. For solid
LB medium, add 15 g of agar.
4. Antibiotics stocks. Ampicillin, carbenicillin, and kanamycin are
dissolved in ddH 2 O at a concentration of 100 mg/mL for
ampicillin and 50 mg/mL for carbenicillin and kanamycin.
Chloramphenicol is dissolved in EtOH at a concentration of
50 mg/mL.
5. Organic solvents for carotenoid and apocarotenoid extraction
and analysis. Acetone, MeOH, CHCl 2 , CHCl 3 .
6. Specific oligonucleotides for cloning in an expression vector
the CCD to be characterized.
7. Expression vectors. Most frequently used expression vectors
are shown in Table 2.
8. 20% arabinose dissolved in ddH 2 O and sterilized by filtration.
9. 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) dissolved
in ddH 2 O and sterilized by filtration.
10. Nitrogen.
11. Vials and columns for HPLC and GC analyses.
Table 1
Plasmids for carotenoids production in E. coli
Plasmid name Genes present
Carotenoid
produced
pAC-PHYTipi Contains crtE, crtB, and idi genes of Erwinia herbicola (Pantoea
agglomerans) Eho10
Phytoene
pAC-ZETAipi Contains crtE, crtB, and idi genes of Erwinia herbicola (Pantoea
agglomerans) Eho10, and crtP gene of Synechococcus PCC7942
ζ-Carotene
pAC-NEUR
Contains the Erwinia herbicola (Pantoea agglomerans) Eho10 genes
crtE and crtB, and the Rhodobacter capsulatus crtI gene
Neurosporene
pAC-LYCipi
Contains crtE, crtB, crtI, and idi genes of Erwinia herbicola (Pantoea
agglomerans) Eho10
Lycopene
pAC-DELTA
Contains crtE, crtB, and crtI genes of Erwinia herbicola (Pantoea
agglomerans) Eho10, together with the lcyE gene of Arabidopsis
thaliana
δ-Carotene
pAC-BETAipi Contains ctrE, crtB, crtI, crtY, and idi genes of Erwinia herbicola
(Pantoea agglomerans) Eho10
β-Carotene
pAC-ZEAXipi Contains crtE, idi, crtI, crtY, crtB, and crtZ genes of Erwinia herbicola
(Pantoea agglomerans) Eho10
Zeaxanthin
pAC-VIOL
Contains crtE, idi, crtI, crtY, crtB, and crtZ genes of Erwinia herbicola
(Pantoea agglomerans) Eho10 and ZEP from Arabidopsis thaliana
Violaxanthin
All the plasmids are low copy bacterial cloning vectors and provide chloramphenicol resistance
Determination of In Vitro and In Vivo Activities of Plant Carotenoid. . .
65