pGTEVΔNIb with Eco81I and ApaI, and follow the same
procedure (Fig. 3).
8. Mix the appropriate amounts of the DNA fragments to set up a
DNA ligation reaction with T4 DNA ligase for 1 h at 22
C.
9. Purify the products of ligation reaction using a silica gel column
and employ them to electroporate E. coli DH5α.
10. Select the transformed bacteria in the LB agar plates containing
kanamycin.
11. Miniprep the plasmids from some colonies and analyze them
by electrophoresis in a 1% agarose gel.
12. Choose a plasmid with the expected migration (see Note 7) and
confirm the incorporation of the desired DNA fragment first by
a restriction analysis and then, if necessary, by sequencing.
3.2 Transformation
of A. tumefaciens
1. Using the pGTEVΔNIb derivative that contains the TEV
recombinant clone, electroporate an A. tumefaciens C58C1
clone that harbors helper plasmid pCLEAN-S48 [15] (see
Note 8).
2. Select the transformed bacteria in the LB agar plates containing
rifampicin, tetracycline and kanamycin. Incubate plates for
5–7 days at 28
C (see Note 9).
3.3 Plant
Agroinoculation
1. Pick an A. tumefaciens colony from the plate with three antibiotics and inoculate 10 mL of the liquid LB media containing
kanamycin (see Note 10).
2. Grow the liquid culture for approximately 24 h at 28
C with
shaking (200 revolutions per minute, rpm).
3. Measure the optic density of the culture at 600 nm and harvest
bacteria at the exponential grow rate (see Note 11).
4. Based on the actual optic density at which A. tumefaciens are
harvested, spin down an aliquot of the bacterial culture to
prepare 4.5 mL of cells in agroinoculation media (see Note 12).
5. Induce the A. tumefaciens culture for 3 h at 28
C with shaking
(200 rpm).
6. Use a 1-mL needleless syringe to infiltrate the induced
A. tumefaciens culture into the abaxial side of an
N. benthamiana plant leaf that expresses TEV NIb. The middle
leaves of the plant (youngest among those fully expanded)
usually give the best results (see Note 13).
3.4 Carotenoid
Production
1. Cultivate plants in a growth chamber at 25
C with a common
day-night cycle.
2. Symptoms of infection should be visible in young tissue around
1 week after inoculation (see Note 14).
Potyvirus Vectors to Produce Carotenoids
309
procedure (Fig. 3).
8. Mix the appropriate amounts of the DNA fragments to set up a
DNA ligation reaction with T4 DNA ligase for 1 h at 22
C.
9. Purify the products of ligation reaction using a silica gel column
and employ them to electroporate E. coli DH5α.
10. Select the transformed bacteria in the LB agar plates containing
kanamycin.
11. Miniprep the plasmids from some colonies and analyze them
by electrophoresis in a 1% agarose gel.
12. Choose a plasmid with the expected migration (see Note 7) and
confirm the incorporation of the desired DNA fragment first by
a restriction analysis and then, if necessary, by sequencing.
3.2 Transformation
of A. tumefaciens
1. Using the pGTEVΔNIb derivative that contains the TEV
recombinant clone, electroporate an A. tumefaciens C58C1
clone that harbors helper plasmid pCLEAN-S48 [15] (see
Note 8).
2. Select the transformed bacteria in the LB agar plates containing
rifampicin, tetracycline and kanamycin. Incubate plates for
5–7 days at 28
C (see Note 9).
3.3 Plant
Agroinoculation
1. Pick an A. tumefaciens colony from the plate with three antibiotics and inoculate 10 mL of the liquid LB media containing
kanamycin (see Note 10).
2. Grow the liquid culture for approximately 24 h at 28
C with
shaking (200 revolutions per minute, rpm).
3. Measure the optic density of the culture at 600 nm and harvest
bacteria at the exponential grow rate (see Note 11).
4. Based on the actual optic density at which A. tumefaciens are
harvested, spin down an aliquot of the bacterial culture to
prepare 4.5 mL of cells in agroinoculation media (see Note 12).
5. Induce the A. tumefaciens culture for 3 h at 28
C with shaking
(200 rpm).
6. Use a 1-mL needleless syringe to infiltrate the induced
A. tumefaciens culture into the abaxial side of an
N. benthamiana plant leaf that expresses TEV NIb. The middle
leaves of the plant (youngest among those fully expanded)
usually give the best results (see Note 13).
3.4 Carotenoid
Production
1. Cultivate plants in a growth chamber at 25
C with a common
day-night cycle.
2. Symptoms of infection should be visible in young tissue around
1 week after inoculation (see Note 14).
Potyvirus Vectors to Produce Carotenoids
309