using primers designed according to In-Fusion HD Cloning
Plus kit to insert into the vector as a single PCR fragment.
BirA* is from Roux et al. 2012.
3. Figure 1a is a representation of pDB vector multiple cloning
site. pDB is derived from pTA7002 with a DEX-inducible
GVG chimeric transcription factor, consisting of the
DNA-binding domain GAL4, the transactivating domain of
the herpes viral protein VP16, and the hormone-binding
domain of the rat glucocorticoid receptor (GR), under control
of the cauliflower mosaic virus 35S promoter [19]. The pDB
vector includes a modified multiple cloning site that allows
cloning in frame with an HA epitope tag to detect the protein
of interest (Fig. 1a).
4. We digested pDB with StuI and cloned our gene of interest
with BirA* in frame with the HA epitope tag.
5. Using an inducible promoter allows for control of the biotinylation period as well as the plant developmental stage of interest. Conditional expression also limits negative effects from
expressing deleterious proteins such as pathogenic effectors.
6. Using BirA* as a negative control excludes all proteins that are
being nonspecifically biotinylated by the promiscuous biotin
ligase. This allows for enrichment of bait-specific targets to be
identified.
7. We used homozygous transgenic plants, but heterozygous lines
can also be used for BioID.
8. Using WT plants as a negative control allows for exclusion of
endogenously biotinylated proteins.
9. Use only glass syringe or glass pipette to take out formic acid to
prevent peaks from plastic contamination in mass spectrometry.
10. We have used In-Fusion HD Cloning Plus kit with success.
11. Avoid contamination between different constructs by using
fresh gloves and beaker in between different constructs and
keeping the dipped pots in separate trays.
12. Spray the plants in a fume hood with gloves and lab coat.
13. Spray the seedlings thoroughly, making sure to spray all the
edges and clumps of seeds. If the seeds haven’t germinated by
day 3, then wait till day 5. After 3 days of spraying, spray any
seedlings that look resistant (green) thoroughly to confirm
resistance. All the seeds in the Col-0 negative control pot
should turn yellow and die. After seedlings have been sprayed
4–5 times, transfer resistant seedlings from the transgenic lines
to a fresh pot of soil. Transgenic rate is usually between 2 and
3% of total seeds.
14. Use goggles as the solution may spray.
438
Madiha Khan et al.
Plus kit to insert into the vector as a single PCR fragment.
BirA* is from Roux et al. 2012.
3. Figure 1a is a representation of pDB vector multiple cloning
site. pDB is derived from pTA7002 with a DEX-inducible
GVG chimeric transcription factor, consisting of the
DNA-binding domain GAL4, the transactivating domain of
the herpes viral protein VP16, and the hormone-binding
domain of the rat glucocorticoid receptor (GR), under control
of the cauliflower mosaic virus 35S promoter [19]. The pDB
vector includes a modified multiple cloning site that allows
cloning in frame with an HA epitope tag to detect the protein
of interest (Fig. 1a).
4. We digested pDB with StuI and cloned our gene of interest
with BirA* in frame with the HA epitope tag.
5. Using an inducible promoter allows for control of the biotinylation period as well as the plant developmental stage of interest. Conditional expression also limits negative effects from
expressing deleterious proteins such as pathogenic effectors.
6. Using BirA* as a negative control excludes all proteins that are
being nonspecifically biotinylated by the promiscuous biotin
ligase. This allows for enrichment of bait-specific targets to be
identified.
7. We used homozygous transgenic plants, but heterozygous lines
can also be used for BioID.
8. Using WT plants as a negative control allows for exclusion of
endogenously biotinylated proteins.
9. Use only glass syringe or glass pipette to take out formic acid to
prevent peaks from plastic contamination in mass spectrometry.
10. We have used In-Fusion HD Cloning Plus kit with success.
11. Avoid contamination between different constructs by using
fresh gloves and beaker in between different constructs and
keeping the dipped pots in separate trays.
12. Spray the plants in a fume hood with gloves and lab coat.
13. Spray the seedlings thoroughly, making sure to spray all the
edges and clumps of seeds. If the seeds haven’t germinated by
day 3, then wait till day 5. After 3 days of spraying, spray any
seedlings that look resistant (green) thoroughly to confirm
resistance. All the seeds in the Col-0 negative control pot
should turn yellow and die. After seedlings have been sprayed
4–5 times, transfer resistant seedlings from the transgenic lines
to a fresh pot of soil. Transgenic rate is usually between 2 and
3% of total seeds.
14. Use goggles as the solution may spray.
438
Madiha Khan et al.