solution by autoclaving. Prepare the working solution by adding 1.5 μL of the 100 mM acetosyringone stock per 1 mL of
the final solution to reach an acetosyringone final concentration of 150 μM.
3 Methods
3.1 Construction
of the Recombinant
Virus
To build a recombinant TEV that expresses one carotenogenic
protein or more, the cDNAs corresponding to these proteins are
first assembled in intermediate plasmids pTV1 or pTV3, depending
on these heterologous proteins are intended to be expressed from
the amino terminal end or internally in the viral polyprotein,
respectively (Fig. 1) (see also Note 2). The combination of both
expression positions is possible.
1. Obtain the cDNA or cDNAs corresponding to the recombinant proteins with the appropriate NIaPro cleavage sites
(Fig. 2) by PCR (see Note 3) or gene synthesis. Flank these
cDNAs by appropriate homologous sequences to assemble
them between positions 454 and 455 of pTV1, or between
474 and 1978 of pTV3 (see Note 4).
2. Amplify by PCR the DNAs corresponding to pTV1 or/and
pTV3 opened between positions 454 and 455, and between
474
and
1978,
respectively.
Use
primers
5
0 -
GGCTATCGTTCGTAAATGG-3
0
and
5
0 -ATGGCACTCATCTTTGGC-3
0 to open pTV1, and primers
5
0 -CTTCTCCCCTTGCGAGTAC-3
0
and
5
0 -ACGACTGAGAATCTTTATTTTCAG-3
0 to open pTV3.
3. Mix all the DNA fragments at a vector–insert 1:3 molar ratio
and perform the Gibson assembly reaction for 1 h at 50
C [16]
(see also Note 5).
4. Purify the DNA from the assembly reaction using silica gel
columns and use it to electroporate Escherichia coli DH5α.
5. Select the transformed E. coli clones in the LB agar plates
containing chloramphenicol.
6. Miniprep the plasmids from some of the colonies and analyze
them by electrophoresis in 1% agarose gel to select one with
the expected migration according to the inserted cDNAs (see
Note 6).
7. For the expression from the amino terminal end of the polyprotein, digest both the pTV1 derivative and pGTEVΔNIb
with NotI and PteI, separate the DNA fragments by electrophoresis in 1% agarose gel and elute the appropriate fragments
from the gel using silica gel columns. For the expression from
inside the viral polyprotein, digest the pTV3 derivative and
308
Jose ´ -Antonio Daro ` s
the final solution to reach an acetosyringone final concentration of 150 μM.
3 Methods
3.1 Construction
of the Recombinant
Virus
To build a recombinant TEV that expresses one carotenogenic
protein or more, the cDNAs corresponding to these proteins are
first assembled in intermediate plasmids pTV1 or pTV3, depending
on these heterologous proteins are intended to be expressed from
the amino terminal end or internally in the viral polyprotein,
respectively (Fig. 1) (see also Note 2). The combination of both
expression positions is possible.
1. Obtain the cDNA or cDNAs corresponding to the recombinant proteins with the appropriate NIaPro cleavage sites
(Fig. 2) by PCR (see Note 3) or gene synthesis. Flank these
cDNAs by appropriate homologous sequences to assemble
them between positions 454 and 455 of pTV1, or between
474 and 1978 of pTV3 (see Note 4).
2. Amplify by PCR the DNAs corresponding to pTV1 or/and
pTV3 opened between positions 454 and 455, and between
474
and
1978,
respectively.
Use
primers
5
0 -
GGCTATCGTTCGTAAATGG-3
0
and
5
0 -ATGGCACTCATCTTTGGC-3
0 to open pTV1, and primers
5
0 -CTTCTCCCCTTGCGAGTAC-3
0
and
5
0 -ACGACTGAGAATCTTTATTTTCAG-3
0 to open pTV3.
3. Mix all the DNA fragments at a vector–insert 1:3 molar ratio
and perform the Gibson assembly reaction for 1 h at 50
C [16]
(see also Note 5).
4. Purify the DNA from the assembly reaction using silica gel
columns and use it to electroporate Escherichia coli DH5α.
5. Select the transformed E. coli clones in the LB agar plates
containing chloramphenicol.
6. Miniprep the plasmids from some of the colonies and analyze
them by electrophoresis in 1% agarose gel to select one with
the expected migration according to the inserted cDNAs (see
Note 6).
7. For the expression from the amino terminal end of the polyprotein, digest both the pTV1 derivative and pGTEVΔNIb
with NotI and PteI, separate the DNA fragments by electrophoresis in 1% agarose gel and elute the appropriate fragments
from the gel using silica gel columns. For the expression from
inside the viral polyprotein, digest the pTV3 derivative and
308
Jose ´ -Antonio Daro ` s