 |
 |
 |
 |
|
 |
 |
7
The binary destination vector was constructed by subcloning the Gateway conversion cassette
C.1 (Invitrogen) into the filled-in EcoRI-HindIII sites of the promoterless T-DNA region of pBIN19.
The resulting Gateway destination vector, designated pBIN-GW, has the following structure in its T-
DNA region: T-DNA right border-NOS terminator<-NPTII<-35S promoter-attR1->CAT->ccdB-
>attR2-T-DNA left border. This vector has no regulatory sequences for expression of cloned genes
and, thus, is useful for producing native levels and patterns of gene expression.
Using a similar strategy, the pMN20 activation tagging plasmid (Weigel et al. 2000) was
converted to a Gateway vector by subcloning the Gateway conversion cassette C.1 into the filled-in
HindIII site of the T-DNA region of pMN20. The resulting plasmid, pMN-GW, has the following
structure of its T-DNA region: T-DNA right border-(35S enhancer)
4
-attR1->CAT->ccdB->attR2-35S
promoter-NPTII-NOS terminator-T-DNA left border. This vector has tetramerized CaMV 35S enhancers
in its T-DNA region (Weigel et al. 2000) and, thus, is useful for producing elevated levels of gene
expression while retaining native expression patterns.
Note that pMN20-based vectors should be prepared from fresh bacterial stocks and used
immediately after transferring them to Agrobacterium because they tend to lose some copies of their 35S
enhancers due to recombination in E. coli or Agrobacterium when stored at 4°C (Weigel et al. 2000).
Also note that unrecombined destination vectors should be propagated in the ccdB-resistant DB3.1 strain
of E. coli (Invitrogen) whereas, following Gateway recombination, the recombinant clones should be
propagated in the ccdB-sensitive bacterial strains such as DH5
or DH10B (see description of
pDONR207 above for more details).
b. LR reaction and selection and identification of recombinant clones
The tagged gene is transferred to the binary destination vector by in vitro recombination
between the attL1 and attL2 sequences that flank the TT-PCR product in the pDONR vector (see
above) and the attR1 and attR2 sequences, respectively, of the destination vector (Landy 1989, see also
www.invitrogen.com). This attL x attR recombination is mediated by the LR reaction (Invitrogen) and
produces the attB1 and attB2 sequences that flank the tagged gene within the binary vector.
LR reaction mixture
LR reaction conditions
200 ng pDONR construct
overnight incubation at 25ºC
200 ng 1:1 w/w mixture of pBIN-GW and pMN-GW*
0.5 µl topoisomerase I (10 U/µl)
2 µl 5x LR Clonase reaction buffer
2 µl LR Clonase (Invitrogen)
TE buffer (pH 8.0) to total volume of 10 µl
*Note that unrecombined destination
vectors are toxic to most bacterial
strains and should be propagated in the
DB3.1 strain of E. coli (Invitrogen) in the
presence of chloramphenicol and
kanamycin (pBIN-GW) or spectinomycin
(pMN-GW)
Add 1 µl Proteinase K (2 µg/µl) and incubate for 10 minutes at 37ºC. Then, transform 2µl of
the reaction mixture into 100 µl competent cells of the E. coli strain DH5
or DH10B and plate one
half of the transformation mixture on LB agar supplemented with 50 µg/ml kanamycin to select for
pBIN-GW recombinants and the other half — on LB agar supplemented with 100 µg/ml
spectinomycin to select for pMN-GW recombinants.
Pick 2 colonies per construct and analyze each by PCR, using P1 and P4 primers, for the
presence of the TT-PCR product. In our experiments, the efficiency of the recombination of the TT-
PCR products from pDONR into the binary destination vector is 90-100%.
III. Production of transgenic Arabidopsis expressing the tagged genes
1. Introduction of binary constructs into Agrobacterium
(i) Grow Agrobacterium tumefaciens strain GV3101 containing the pMP90 helper plasmid
(carrying gentamycin resistance) in 5 ml of LB medium overnight at 28°C.
|
|
|
|
|
|
|