7. Growth media with antibiotics: prepare the final media by
adding 1 μL of the corresponding antibiotic stock per 1 mL
of medium under sterile conditions. The final concentrations
are 34 μg/mL chloramphenicol, 50 μg/mL rifampicin,
7.5 μg/mL tetracycline (see Note 1), and 50 μg/mL
kanamycin.
2.3 Electrophoresis
1. TAE electrophoresis buffer: 40 mM Tris, 20 mM sodium acetate, 1 mM sodium ethylenediaminetetraacetate (EDTA),
pH 7.2. Prepare (10Â) concentrated by weighing 97 g of
Tris, 54 g of sodium acetate (·3H 2 O) and 7 g of Na 2 EDTA·2H 2 O, and dissolve in water. Adjust pH to 7.2 with acetic
acid and bring to a final volume of 1 L with water. There is no
need to use this solution under sterile conditions, although
autoclaving is a common practice to improve conservation at
room temperature.
2. 1% agarose electrophoresis gel: weigh 1 g of agarose and dissolve in 100 mL (Â1) of TAE buffer by heating. Adjust the
solution to approximately 65
C and cast the agarose gel.
Melted agarose can be stored for a few days at 65
C.
2.4 Agrobacterium
tumefaciens Induction
Media
1. Acetosyringone stock solution (100 mM): weigh 0.59 g of
acetosyringone and dissolve in 30 mL of dimethyl sulfoxide.
Prepare aliquots and store them frozen at À20
C.
2. A. tumefaciens induction solution: 10 mM 2-(N-morpholino)
ethanesulfonic acid (MES)-NaOH, pH 5.6, 10 mM MgCl 2 ,
150 μM acetosyringone. Weigh 1.07 g of MES·H 2 O and
1.02 g of MgCl 2 ·6H 2 O, and dissolve with water. Adjust pH
to 5.6 with 0.1 M NaOH and bring to 500 mL. Sterilize the
Fig. 4 Pictures of Nicotiana benthamiana leaves infected with TEV recombinant clones that express carotenogenic proteins. (A) Expression of Pantoea ananatis phytoene synthase (crtB). Upper noninoculated leaf
photographed at 7 dpi. (B) Expression of P. ananatis crtE: geranylgeranyl pyrophosphate synthase (crtE),
crtB and phytoene desaturase (crtI). Agroinoculated leaf photographed at 6 dpi
Potyvirus Vectors to Produce Carotenoids
307
adding 1 μL of the corresponding antibiotic stock per 1 mL
of medium under sterile conditions. The final concentrations
are 34 μg/mL chloramphenicol, 50 μg/mL rifampicin,
7.5 μg/mL tetracycline (see Note 1), and 50 μg/mL
kanamycin.
2.3 Electrophoresis
1. TAE electrophoresis buffer: 40 mM Tris, 20 mM sodium acetate, 1 mM sodium ethylenediaminetetraacetate (EDTA),
pH 7.2. Prepare (10Â) concentrated by weighing 97 g of
Tris, 54 g of sodium acetate (·3H 2 O) and 7 g of Na 2 EDTA·2H 2 O, and dissolve in water. Adjust pH to 7.2 with acetic
acid and bring to a final volume of 1 L with water. There is no
need to use this solution under sterile conditions, although
autoclaving is a common practice to improve conservation at
room temperature.
2. 1% agarose electrophoresis gel: weigh 1 g of agarose and dissolve in 100 mL (Â1) of TAE buffer by heating. Adjust the
solution to approximately 65
C and cast the agarose gel.
Melted agarose can be stored for a few days at 65
C.
2.4 Agrobacterium
tumefaciens Induction
Media
1. Acetosyringone stock solution (100 mM): weigh 0.59 g of
acetosyringone and dissolve in 30 mL of dimethyl sulfoxide.
Prepare aliquots and store them frozen at À20
C.
2. A. tumefaciens induction solution: 10 mM 2-(N-morpholino)
ethanesulfonic acid (MES)-NaOH, pH 5.6, 10 mM MgCl 2 ,
150 μM acetosyringone. Weigh 1.07 g of MES·H 2 O and
1.02 g of MgCl 2 ·6H 2 O, and dissolve with water. Adjust pH
to 5.6 with 0.1 M NaOH and bring to 500 mL. Sterilize the
Fig. 4 Pictures of Nicotiana benthamiana leaves infected with TEV recombinant clones that express carotenogenic proteins. (A) Expression of Pantoea ananatis phytoene synthase (crtB). Upper noninoculated leaf
photographed at 7 dpi. (B) Expression of P. ananatis crtE: geranylgeranyl pyrophosphate synthase (crtE),
crtB and phytoene desaturase (crtI). Agroinoculated leaf photographed at 6 dpi
Potyvirus Vectors to Produce Carotenoids
307