Double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) used in this study were quantified by Quant-iT dsDNA Broad-Range Assay Kit (Life Technologies) and ssDNA Assay Kit (Life Technologies), respectively. 10 μl of sample or standard DNA was added to 0.6 ml Flat Cap PCR Tube (Sorenson BioScience), followed by adding 190 μl of working solution, composited by 99.5%
(v/v) of buffer and 0.5% (v/v) of fluorescent dye from the assay kits. The sample and standard reactions were then mixed by vigorously shaking and incubated for 2 minutes at room temperature. The DNA concentration was measured and calculated by Qubit 2.0 Fluorometer (Life Technologies).
1.2. Plasmid DNA extraction from E. coli
The plasmid isolation from E. coli was carried out by alkaline lysis method.
Single colony of E. coli was inoculated in a glass tube containing 3 ml Luria-Bertani (LB; which consists of 10 g/l tryptone, 5 g/l yeast extract, and 10 g/l NaCl, pH 7.0) and the appropriate antibiotics at 37℃ with shaking overnight. The cell pellet was collected by centrifugation at 6,000 x g for 2 minutes. To resuspend the cells, 300 μl of solution I (25 mM Tris-HCl, pH 8; 10 mM EDTA; 10 μg/ml RNase (Sigma R-4642) was added to the cells with vigorous shaking. Then, 300 μl of solution II (0.2 N NaOH; 1% SDS) was added and mixed gently by inverting the tube and the mixture was incubated at room temperature for 5 minutes to lyse the cells completely.
After incubation, 300 μl ice-cold solution III (3 M potassium acetate, pH 5.2) was added and mixed immediately by inverting the tube to precipitate the genomic DNA
and proteins. The solution was incubated on ice for another 5 minutes to precipitate potassium dodecyl sulfate to improve the purity of plasmid DNA. Centrifugation was performed at 18,000 x g for 10 minutes at 4℃ and the supernatant containing plasmid DNA was transferred to a new centrifuge tube. The plasmid DNA was further precipitated by adding 0.6 volume (540 μl) of isopropanol and centrifuging at 18,000 x g for 30 minutes at 4℃. The supernatant was discarded, and the plasmid DNA pellet was washed once by adding 1 ml of 70% ethanol and centrifuging at 18,000 x g for 10 minutes at 4℃ to remove the residual isopropanol. The supernatant was discarded and the plasmid DNA pellet was allowed air-dry for 10 minutes to evaporate ethanol. The plasmid DNA was redissolved either in deionized water for electroporation transformation or in TE (10 mM Tris-HCl, pH 8.0; 1 mM EDTA) buffer for chemical transformation, PCR analysis, sequencing, and endonuclease reaction.
1.3. Polymerase chain reaction (PCR)
In this study, five different DNA polymerases (or premix reagents) were used in PCR of different purposes. For routine PCR, Taq DNA Polymerase Master Mix Red (Ampliqon) was used. Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific) was applied in cloning. Expand High Fidelity PCR System (Roche) was used to synthesize DIG-labeled DNA probe. Advantage 2 Polymerase Mix (Clontech) was applied in long distance PCR for constructing Gal4 activation domain-fused cDNA library to identify the positive yeast two-hybrid clones of the library. iQ SYBR Green Supermix (Bio-Rad) was used in quantification PCR (described in the Material and Methods section 2.4, reverse transcriptase quantification polymerase chain reaction). Except the quantification PCR, all other PCR reaction mixtures containing
1x PCR buffer was supplied by the respective companies. In general, 1.5 mM of magnesium (II) chloride, 0.4 mM of dNTP (each), 200 nM of primers (each), and 1 ng/μl of genomic DNA templates or 1 pg/μl of plasmid DNA templates were used.
PCR conditions were shown in Supplementary Table S2.
1.4. DNA purification
In aqueous solution such as PCR product, DNA was precipitated by adding 1/10 volume of 3 M sodium acetate (pH 5.2, adjusted by acetic acid) followed by adding 2 volumes of absolute ethanol. Centrifuging at 18,000 x g for 30 minutes at 4℃ to pellet the DNA and the supernatant was removed by pipet tip, and the pellet was air-dried for 10 minutes to evaporate the residue ethanol. The DNA pellet is redissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). If the targeted nucleotide fragment is isolated from a polynucleotide mixture, agarose gel electrophoresis coupled with gel extraction is applied. After electrophoresis, the agarose gel containing target DNA fragment was excised and transferred to a microcentrifuge tube, and the DNA was extracted from the gel with Zymoclean Gel DNA Recovery Kit (Zymo Research). Appropriate volume (100 μl for every 100 mg of agarose gel) of ADB Buffer was added to the tube and incubated at 45℃ until the agarose gel dissolved in the buffer (approximate 15 minutes). The solution was loaded onto the Zymo-Spin I Column, and the column was centrifuged at 18,000 x g for 30 seconds, and the flow through was discarded. The column was then washed by 200 μl of DNA Wash Buffer twice under the same centrifugation condition. After wash step, the DNA was eluted by 6 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). These DNA purification procedures were taken to obtain a single purified DNA fragment for many downstream applications in this study, such as PCR, splicing by overlap
extension PCR (SOEing PCR), endonuclease reaction, sequencing, ligation, and in vitro LR recombination.
1.5. Cloning by restriction-ligation method
pK18mobsacB-rolA, B, C, and D vectors for deleting respective rol genes, pGBKT7-rolA, B, C, and D vectors for constructing bait vector for yeast two-hybrid, and pET-21d-rolB and rolC for in vitro phosphatase activity assay in this thesis were cloned by restriction-ligation method. PCR product was purified by agarose gel electrophoresis, and 25 μl of eluted DNA was incubated with 1 volume of 2x Taq DNA Polymerase Master Mix Red (Ampliqon) at 72℃ for 10 minutes to incorporate adenine bases to the both ends of PCR products. The products were further purified by isopropanol precipitation and resuspended in 10 μl of tris buffer. Then, 2 μl of product was added to a microcentrifuge tube containing 1 μl of yT&A cloning vector (Yeatern), 1 μl of T4 DNA ligase buffer (New England Biolab), 5 μl of deionized water, and 1 μl of T4 DNA ligase (New England Biolab). The reaction was carried out for 2 hours at room temperature to create cloning vector, and the result plasmid was transform into E. coli by heat shock mentioned below. The cloning and expressing plasmids were extracted and incubated with restriction enzymes respectively, and the fragments were purified by agarose gel electrophoresis.
Afterward, 3:1 molar ratio of insert:vector with the total amount 100 ng of DNA were combined into a microcentrifuge tube containing 1 μl of T4 DNA ligase and 1 μl of buffer (New England Biolab), and then deionized water was added up to 10 μl. The reaction was taken for 2 hours at room temperature followed by E. coli transformation to obtain expression vector.
1.6. Cloning by Gateway system
For transcriptional fusion, pTCrolB and pTCrolC, with yfp driven respectively by rolB and rolC native promoters. For translational fusion, pTLrolB and pTLrolC, yfp-rolB and yfp-rolC driven by respective native promoters, were constructed by Gateway Technology (Life Technologies). 1 μl DNA solution containing 1 fmole purified plasmid or DNA fragment was added to a microcentrifuge tube containing 1 μl of deionized water, 0.5 μl of Salt Solution (Life Technologies), and 0.5 μl of pCR8/
GW/TOPO TA Cloning Vector (Life Technologies). The reaction was carried out at room temperature for 5 minutes. 2 μl of the reaction product was used for E. coli transformation to create entry clone. The transformation procedure was presented in the Materials and Methods section 4.1, E. coli transformation by heat shock. Then, the entry vector and the destination vector containing Gateway fragment were extracted, and 1:1 molar ratio of these two plasmids with the total 75 ng DNA were added into a microcentrifuge tube containing 0.5 μl of LR Clonase II Enzyme Mix (Life Technologies). The reaction was carried out at room temperature for an hour.
Afterward, 0.25 μl of protease K (Life Technologies) was added to inactivate the reaction by incubating at 37℃ for 10 minutes. 2 μl of the resulting mixture was used for E. coli transformation to obtain expression clones.
1.7. Total DNA isolation from A. rhizogenes
Agrobacterium total DNA was extracted with Wizard Genomic Purification Kit (Promega). One colony of A. rhizogenes was cultured in 3 ml yeast extract broth (YEB; which consists of 5 g/l beef extract, 1 g/l yeast extract, 5 g/l peptone, 5 g/l sucrose, and 0.49 g/l magnesium chloride heptahydrate) containing the appropriate antibiotics for 48 hours. One milliliter of cells (OD600=1) were added to a
microcentrifuge tube. The cells were pelleted by centrifugation at 18,000 x g for 2 minutes. 600 μl of Nuclei Lysis Solution (Promega) was added to resuspend the cells by gently pipetting up and down, and the cells were incubated at 80℃ for 5 minutes for lysis. The lysate was cooled to room temperature. 1 μl of RNase A (Sigma R-4642) was added and mixed gently by inverting the tube, and the cell lysate was incubated at 37℃ for 30 minutes to reduce RNA contaminations. Then, 200 μl of Protein Precipitation Solution (Promega) was added to the RNase A-treated cell lysate and the sample was mixed by vortex vigorously for 20 seconds followed by incubating on ice for 5 minutes. Afterward, centrifugation at 18,000 x g for 3 minutes and the supernatant containing the genomic DNA was transferred to a new microcentrifuge tube. The genomic DNA was further purified by isopropanol precipitation and 70% ethanol wash. The air-dried genomic DNA pellet was rehydrated in TE buffer by incubating at 60℃ for 1 hours, followed by incubating in 4℃ refrigerator overnight. The A. rhizogenes genomic DNA was ready to be applied to cloning, PCR analysis, DIG-probe synthesis, and endonuclease treatment for Southern blot.
1.8. Plasmid DNA extraction from yeast
A single yeast colony was inoculated into 0.5 ml of the SD (synthetic defined) medium (6.7 g/l yeast nitrogen base without amino acids, 20 g/l dextrose) with 50 ppm kanamycin and appropriate amino acid supplement overnight at 30℃ with shaking. The cells were transferred to a microcentrifuge tube and then pelleted by centrifugation at 18,000 x g for 30 seconds. The supernatant was removed and the pellet was resuspended in 50 μl TE buffer. 2 μl of 5 U/μl Zymolyase solution (Zymo Research) was added and incubated at 37℃ for an hour with vigorous shaking to lyse
the yeast cell walls. Then, 20 μl of 10% sodium dodecyl sulfate (SDS) was added to cells and vortexed vigorously to lyse cells. To extract the DNA, the sample volume was brought to 200 μl with deionized water. Then, 200 μl of buffered phenol (pH 8.0) was added to the sample and mixed by vigorously vortexing. Centrifugation was carried out at 18,000 x g for 10 min and the upper aqueous phase was transferred to a new microcentrifuge tube. These phenol extraction steps were repeated until the interphase between aqueous solution and phenol was clean. To remove the phenol contamination, 200 μl of chloroform:isoamyl alcohol (24:1) was added, mixed and centrifuged as phenol extraction, and the final aqueous solution containing yeast DNA was further purified by isopropanol precipitation. The DNA pellet was redissolved in TE buffer for downstream PCR analysis or E. coli transformation to recover plasmid DNA.