11. LB liquid medium: 10 g Tryptone, 10 g NaCl, and 5 g yeast
extract per 1 L, pH 7.0.
12. LB agar plates (prepared using LB liquid medium with the
addition of 15 g/L agar) containing 50 μg/mL kanamycin or
100 μg/mL spectinomycin, respectively.
13. LB liquid medium containing 50 μg/mL kanamycin or
100 μg/mL spectinomycin, respectively.
14. LB agar plates (see Subheading 2.1, item 12) containing
100 μg/mL spectinomycin and 50 μg/mL rifampicin.
15. LB liquid medium containing 100 μg/mL spectinomycin and
50 μg/mL rifampicin.
16. 37
C Incubator.
17. 37
C Shaking incubator.
18. Plasmid miniprep kit.
19. PCR purification kit with a size exclusion limit !50 bp (i.e.,
smaller DNA fragments are removed).
20. 28
C Incubator.
21. 28
C Shaking incubator.
22. Glycerol, sterilized by autoclaving.
23. DNA oligonucleotide primers DONRfor (5
0 -CTGGCAGTTC
CCTACTCTCG-3
0 ), DONRrev (5
0 -ATGTAACATCAGAGA
TTTTGAGACACG-3
0 ), attB1 adapter (5
0 -GGGGACAAGTT
TGTACAAAAAAGCAGGCT-3
0 ), and attB2 adapter (5
0 -GG
GGACCACTTTGTACAAGAAAGCTGGGT-3
0 ).
2.2 Tagging of the
PVX Genome
1. Synthesized gene fragment provided in a cloning vector and
containing two 20 bp Csy4 cognate recognition sequences
(consisting of the 15 bp stem-loop-forming sequence and 5
upstream bases to maximize protein-RNA affinity [13, 14])
(pMA-RQ.NheI-2xcsy-NheI). The two repeats are separated
by an EcoRI restriction site, and flanked on both sides by
NheI sites (see Note 2) (5
TAGGCAGAATTCGTTCACTGCCGTATAGGCAGGCTAGC-3
0 ; stem-loop-forming sequences in bold, restriction
sites italicized). Ligation into a single restriction site allows
for the easy generation of tandem copies of the two stemloop cassettes.
2. Plasmid pTRA.PVX.mCherry-2A-CP containing a PVX
genome with mCherry translationally fused to the capsid protein via a Foot and mouth disease virus 2A ribosomal “skipping” sequence under the control of a Cauliflower mosaic virus
35S promoter [15].
3. NheI and EcoRI restriction enzymes and corresponding 10Â
buffers.
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David Burnett et al.
extract per 1 L, pH 7.0.
12. LB agar plates (prepared using LB liquid medium with the
addition of 15 g/L agar) containing 50 μg/mL kanamycin or
100 μg/mL spectinomycin, respectively.
13. LB liquid medium containing 50 μg/mL kanamycin or
100 μg/mL spectinomycin, respectively.
14. LB agar plates (see Subheading 2.1, item 12) containing
100 μg/mL spectinomycin and 50 μg/mL rifampicin.
15. LB liquid medium containing 100 μg/mL spectinomycin and
50 μg/mL rifampicin.
16. 37
C Incubator.
17. 37
C Shaking incubator.
18. Plasmid miniprep kit.
19. PCR purification kit with a size exclusion limit !50 bp (i.e.,
smaller DNA fragments are removed).
20. 28
C Incubator.
21. 28
C Shaking incubator.
22. Glycerol, sterilized by autoclaving.
23. DNA oligonucleotide primers DONRfor (5
0 -CTGGCAGTTC
CCTACTCTCG-3
0 ), DONRrev (5
0 -ATGTAACATCAGAGA
TTTTGAGACACG-3
0 ), attB1 adapter (5
0 -GGGGACAAGTT
TGTACAAAAAAGCAGGCT-3
0 ), and attB2 adapter (5
0 -GG
GGACCACTTTGTACAAGAAAGCTGGGT-3
0 ).
2.2 Tagging of the
PVX Genome
1. Synthesized gene fragment provided in a cloning vector and
containing two 20 bp Csy4 cognate recognition sequences
(consisting of the 15 bp stem-loop-forming sequence and 5
upstream bases to maximize protein-RNA affinity [13, 14])
(pMA-RQ.NheI-2xcsy-NheI). The two repeats are separated
by an EcoRI restriction site, and flanked on both sides by
NheI sites (see Note 2) (5
TAGGCAGAATTCGTTCACTGCCGTATAGGCAGGCTAGC-3
0 ; stem-loop-forming sequences in bold, restriction
sites italicized). Ligation into a single restriction site allows
for the easy generation of tandem copies of the two stemloop cassettes.
2. Plasmid pTRA.PVX.mCherry-2A-CP containing a PVX
genome with mCherry translationally fused to the capsid protein via a Foot and mouth disease virus 2A ribosomal “skipping” sequence under the control of a Cauliflower mosaic virus
35S promoter [15].
3. NheI and EcoRI restriction enzymes and corresponding 10Â
buffers.
160
David Burnett et al.