Single colony of E. coli was picked from LB plate, inoculated into 3 ml of LB medium in a glass tube and then cultured overnight. The overnight culture was diluted 1000-fold into a new 3 ml LB medium and then grown at 37℃ until the OD600 reached 0.3-0.5. E. coli cell pellet was harvested by transferring the culture medium to pre-chilled centrifuge tube and centrifuging at 1,500 x g for 5 min at 4℃.
The pellet was resuspended in one-tenth of original volume of ice-cold transformation and storage (TSS) solution (LB broth adding 10% w/v polyethylene glycol 8000 (PEG), 5% dimethyl sulfoxide, and 50 mM magnesium chloride). A 0.1 ml aliquot of cells was mixed with 100 ng of plasmid DNA for transformation, and was incubated on ice for 30 minutes. To transform E. coli, the mixture of cells and plasmid DNA was heat shocked by incubating at 42℃ water-bath for 2 minutes, which was
followed by ice-bath for 5 minutes. Next, 250 μl of super optimal broth with catabolite repression (SOC, which consists of 20 g/l tryptone, 5 g/l yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, and 20 mM glucose) was added to the mixture, and the cells were incubated at 37℃ with shaking at 200 rpm for an hour for recovery. To select the transformants, the cells were spread onto LB agar containing appropriate antibiotics and incubated for 16 hours. The transformants were further confirmed by colony PCR and restriction-map analysis.
4.2. A. rhizogenes transformation by electroporation
A glass culture tube containing 3 ml YEB was inoculated with a single colony of the A. rhizogenes. The cells were grown overnight at 26℃ with shaking. The entire 3 ml overnight grown culture was inoculated into a flask containing 100 ml YEB, and the cells were incubated at 26℃ with shaking until the OD600 of the culture reaches 0.5-0.8. The cells were chilled on ice for 10 minutes and transferred to pre-chilled centrifuge tube. Cell pellets were collected by centrifuging at 10,000 x g for 10 minutes at 4℃. Equal volume of ice-cold sterile deionized water was added to wash the cell twice, and one-tenth volume of ice-cold filter-sterile 10% glycerol was added to wash the cell once. Cells were finally resuspended in 400 μl ice-cold filter-sterile 10% glycerol and were aliquoted every 40 μl into individual microcentrifuge tubes.
The cells could be either frozen by liquid nitrogen and stored at -80℃ or processed electroporation directly. To perform Agrobacterium transformation, 100 ng plasmid DNA dissolved in deionized water was added to 40 μl aliquot of cells and incubated on ice for 30 minutes. After incubation, the cells were transferred to a 0.1 cm MicroPulser Cuvette (Bio-Rad 165-2089) and the electroporation was carried out by MicroPulser Electroporator (Bio-Rad) with built-in Agrobacterium transformation
program. One milliliter of YEB was added to the cuvette to rinse the cells, and the mixture was transferred to a glass culture tube. The tube was incubated at 26℃ for 4 hours with shaking. The cells were selected by plating on the YEB agar plates containing the appropriate antibiotics, and further confirmed by PCR with total DNA extracted by Wizard Genomic DNA Purification Kit (Promega).
4.3. Yeast transformation by lithium acetate (LiAc) mediated method
All media used for yeast culture were supplied with 50 ppm kanamycin. Yeast transformation was performed by YeastMaker Yeast Transformation System 2 (Clontech). 3 ml of yeast peptone dextrose adenine (YPDA, which contains 10 g/l yeast extract, 20 g/l peptone, 20 g/l dextrose, and 20 mg/l L-adenine hemisulfate salt) broth was incubated with a yeast colony at 30℃ for 12 hours. Then, 5 μl of the culture was transferred to a 50 ml of YPDA broth in a 250 ml Hinton flask. The culture was incubated at 30℃ with shaking until the OD600 reached 0.15-0.3, and the cells were pelleted by centrifuging at 1,000 x g for 10 minutes and resuspended in 100 ml of fresh YPDA broth. The culture was incubated at 30℃ until the OD600
reached 0.4-0.5. The cells were pelleted and washed by adding 100 ml of sterile deionized water followed by 3 ml of 1.1 x TE/LiAc (1 x TE/LiAc consists of 10 mM Tris-HCl, 1 mM EDTA, and 100 mM LiAc; pH 7.5), and finally resuspended in 1.2 ml of 1.1 x TE/LiAc solution. To introduce a plasmid into yeast cells, 100 ng of plasmid was added into 50 μl of 1.1 x TE/LiAc suspended cells. 5 μl of YeastMaker Carrier DNA and 500 μl of PEG/LiAc (40% w/v PEG 4000; 1 x TE/LiAc) was added to the cells, mixed and incubated at 30℃ for 30 minutes. After incubation, 20 μl of dimethyl sulfoxide (DMSO) was added to the cells and incubated at 42℃ for 15 minutes. Then, the cell pellet was collected by centrifugation at 18,000 x g for 30
seconds, and the cells were resuspended in 1 ml of 0.9% NaCl and the positive transformants were selected by plating onto SD agar with appropriate amino acid supplements. In addition to single plasmid transformation, library-scale transformation was performed in this study to make protein-protein interaction screening using yeast two-hybrid method. In library-scale transformation, DNA was added to 600 μl of 1.1 x TE/LiAc-suspended cells. 20 μl of YeastMaker Carrier DNA and 2.5 ml of PEG/LiAc were added, and 30℃ incubation was taken for 45 minutes.
Then, 160 μl of DMSO was added before 42℃ incubation for 20 minutes. The cells were pelleted and resuspended in 3 ml YPD Plus Medium, and incubated at 30℃ for 90 minutes with shaking to increase transformation efficiency. The cells were pelleted again and resuspended in 15 ml of 0.9% NaCl. The transformants were selected by plating onto SD agar (150 μl per 15 cm petri dish) with appropriate amino acid supplements.
5. Individual rol genes deficient strains generation