3. Harvest the symptomatic tissue 2 days later for the carotenoid
analysis (Fig. 4).
4 Notes
1. The tetracycline concentration to select pCLEAN-S48 (see
below) is a slightly lower than those normally used with the
plasmids containing tetracycline resistance selection markers.
2. From the amino terminal end of the polyprotein, heterologous
proteins can be targeted to any subcellular location, provided
that they harbor an appropriate signal peptide. From inside the
polyprotein, heterologous proteins can be efficiently targeted
only to the nucleus and the cytosol [12].
3. For the PCR amplifications, the use of a high-fidelity DNA
polymerase, such as Phusion DNA polymerase (Thermo Scientific), is recommended.
4. The use of a program or web tool, such as the NEBuilder
Assembly Tool from New England BioLabs (https://
nebuilder.neb.com/#!/), facilitates the design of the flanking
sequences that allow DNA assembly during the Gibson reaction. Separate the cDNAs corresponding to the different proteins by the À8/+3 cleavage peptides recognized by TEV
NIaPro. To avoid undesired recombinations during the replication of the recombinant virus, while maintaining the same
amino acid sequence (TTENLYFQ/SGT), use different nucleotide sequences. Some examples are found in Fig. 2. Remember that the recombinant protein or block of recombinant
proteins expressed from the amino terminal end of the polyprotein must end with a NIaPro proteolytic site to separate the
last protein from the viral P1 (Fig. 1). If the recombinant
protein or proteins are expressed from inside the polyprotein,
take advantage of the native NIaPro/NIb and NIb/CP cleavage sites that remain in pTV3 for the first and last cleavages.
5. For the Gibson reaction, we successfully used the NEBuilder
HiFi DNA Assembly Master Mix from New England Biolabs.
6. Plasmid sequencing around the region where the insertion
takes place is recommended.
7. Due to the size of the plasmids at this point, do not expect to
observe a clear difference in migration between those containing the recombinant virus and those with the unmodified
TEVΔNIb clone. A restriction analysis and sequencing are
recommended for confirmation purposes.
8. pGTEVΔNIb is a derivative of pCLEAN-G181 (GenBank
accession number EU186083.1) [15] and requires the presence of a helper plasmid of the pSoup series to replicate in
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Jose ´ -Antonio Daro ` s
analysis (Fig. 4).
4 Notes
1. The tetracycline concentration to select pCLEAN-S48 (see
below) is a slightly lower than those normally used with the
plasmids containing tetracycline resistance selection markers.
2. From the amino terminal end of the polyprotein, heterologous
proteins can be targeted to any subcellular location, provided
that they harbor an appropriate signal peptide. From inside the
polyprotein, heterologous proteins can be efficiently targeted
only to the nucleus and the cytosol [12].
3. For the PCR amplifications, the use of a high-fidelity DNA
polymerase, such as Phusion DNA polymerase (Thermo Scientific), is recommended.
4. The use of a program or web tool, such as the NEBuilder
Assembly Tool from New England BioLabs (https://
nebuilder.neb.com/#!/), facilitates the design of the flanking
sequences that allow DNA assembly during the Gibson reaction. Separate the cDNAs corresponding to the different proteins by the À8/+3 cleavage peptides recognized by TEV
NIaPro. To avoid undesired recombinations during the replication of the recombinant virus, while maintaining the same
amino acid sequence (TTENLYFQ/SGT), use different nucleotide sequences. Some examples are found in Fig. 2. Remember that the recombinant protein or block of recombinant
proteins expressed from the amino terminal end of the polyprotein must end with a NIaPro proteolytic site to separate the
last protein from the viral P1 (Fig. 1). If the recombinant
protein or proteins are expressed from inside the polyprotein,
take advantage of the native NIaPro/NIb and NIb/CP cleavage sites that remain in pTV3 for the first and last cleavages.
5. For the Gibson reaction, we successfully used the NEBuilder
HiFi DNA Assembly Master Mix from New England Biolabs.
6. Plasmid sequencing around the region where the insertion
takes place is recommended.
7. Due to the size of the plasmids at this point, do not expect to
observe a clear difference in migration between those containing the recombinant virus and those with the unmodified
TEVΔNIb clone. A restriction analysis and sequencing are
recommended for confirmation purposes.
8. pGTEVΔNIb is a derivative of pCLEAN-G181 (GenBank
accession number EU186083.1) [15] and requires the presence of a helper plasmid of the pSoup series to replicate in
310
Jose ´ -Antonio Daro ` s