| Tryptone | 10.00 g |
| Yeast extract | 5.00 g |
| NaCl | 10.00 g |
| Agar (only for solid medium) | 15.00 g |
a. Weigh the reagents accordingly and add them to a glass beaker and make up the volume to 90% of planned with ddH2O.
b. Dissolve the components in the beaker using a magnetic stirrer
c. Adjust the broth to final volume using ddH2O and adjust the pH to around 7.4
d. Aliquoting and transfer the broth to conical flasks
e. Adding agar if planned to make solid medium
f. Close the mouth of the flasks and place the bottles into the autoclave for sterilization under 121°C for 20 min
g. Cool the sterilized broth without agar to room temperature for use as liquid medium and cool the sterilized broth with agar around 50 °C to make solid medium plates
h. On the ultra-clean Workbench, add sterilized stock solutions of antibiotics accordingly into the broth with agar when it is cooled to around 50 °C (or into the broth without agar before use) and mix well
i. Make solid medium plates
| Tryptone | 5.00 g |
| Yeast extract | 3.00 g |
| CaCl2 | 1.11 g |
| Agar (only for solid medium) | 15.00 g |
a. Prepare stock solution of CaCl2 with concentration of 1 M.
b. Carry out the preliminary work in the same procedure as steps a) – g) for LB medium preparation
c. On the ultra-clean Workbench, add sterilized stock solutions of CaCl2 and antibiotics accordingly into the broth with agar when it is cooled to around 50 °C (or into the broth without agar before use) and mix well.
d. Make solid medium plates
| MS medium powder (with vitamins) (Phyto Technology Laboratories™) | 4.43 g |
| Sucrose | 30.00 g |
| Agar (only for solid medium) | 15.00 g |
Carry out the preliminary work in the same procedure as that for LB medium preparation
Use the method “Golden Gate Assembly (Engler & Marillonnet, S. (2014)” to construct plasmids:
a. Domestication of the Target Sequence
b. Selection of Fusion Sites and Primer Design.
c. PCR Amplification of the Modules.
d. d)Blunt-End Cloning of the Modules.
e. e)Construction of the Destination Vector.
f. Golden Gate Assembly
g. g)Transformation of the Constructs into Competent Cells.
Engler, C., and Marillonnet, S. (2014). Golden Gate cloning. Methods Mol. Biol. 1116, 119–131. doi: 10.1007/978-1-62703-764-8_9
a. Dispense 2 ml of LB liquid medium, add 2 µl of corresponding antibiotics and cap the tube.
b. Prepare three 1.5 ml eppendorf tubes and add 500 µl of culture medium to each
c. Pick monoclones from the Petri dish, one at a time, and inoculate into the eppendorf tube
d. Incubate the bacterial broth for 5 hours to make the broth cloudy
e. Centrifuge at 12000 rpm for 2 minutes to collect the bacteria and aspirate off the supernatant
f. Add 250 µl of ddH2O to the eppendorf tube and blow the bacterial solution
g. Add ddH2O 20 µl, R-primer 10 µl, F-primer 10 µl and the mix 50 µl in 2 eppendorf tubes respectively
h. Each PCR tube is filled with 9 µl of the liquid in the ep tube and 1 µl of the corresponding bacterial solution is added to each tube
i. i)Store the remaining bacterial broth in 4 °C refrigerator (for PCR positive clones, for sequencing or making strains).
j. Place the PCR tube into the PCR machine (Program: User-Cathie-taq-taq1).
a. Add 0.3 g of agarose into 30 ml of 1xTAE stock solution.
b. Heat the 1xTAE solutions with agarose until it becomes transparent.
c. Cool the solutions to about 50 °C.
d. Fix the inner groove glass plate, insert the comb, slowly pour the cooled solution into the glass plate and cool for 20 minutes to solidify
e. Remove the comb, pour in the electrophoresis buffer until plate covered.
b. Mix each 5μl sample with 1 μl Gel Red, transfer dye and plasmids to gel sample holes vertically and slowly with pipetting.
c. Add positive and negative controls as well as plasmids to the corresponding holes.
d. Immediately start the electrophoresis with a voltage of 120 V for 15 minutes
a. Inoculate colony O/N in 2 ml TY + antibiotics at 28 °C shaker. (ABI - 50 KAN & 25 Chlor, gv3101 - 25GEN.)
b. Transfer O/N culture to 200ml TY in a sterile 500ml flask and shake at 250rpm until the OD is 0.3 (4-5 hours)
c. Spin in sterile 50ml screw cap tubes under 4 °C and 5 k rpm for 10, and check to make sure cells are pelleted, if not repeat at higher speed.
d. Aspirate supernatant, resuspend pellet in 20ml ice cold 1mM HEPES pH7 (sterile filtered), respin
e. Repeat d two more times
f. After aspirating, resuspend pellet in 2ml ice cold 10% glycerol (sterile filtered).
g. ASAP dipense in 40 μl aliquots in pre-chilled, sterile eppis, freeze in lN2 and store at -70 °C
Agrobocterium rhizogenes strain A4 is transformed with constructions by electroporation
a. Soak 2-mm wide electroporation cuvettes (BTX®) with lids in 75% ethanol overnight.
b. On the ultra-clean bench, invert electroporation cups and lids on clean absorbent paper to drain for 5 min
c. Place electroporation cuvettes and lids positively on the ultra-clean table and blow-dry them for 5 min
a. Put the electroporation cuvettes on ice
b. Take 100 μl of Agrobacterium competent cells stored at -80 °C out into room temperature.
c. When Agrobacterium competent cells melt into ice-water mixture, add 200 ng of plasmid DNA (the plasmid volume not larger than 6μl, preferably extracted using a kit and solubilized with ddH2O), mix gently, cover the tube, and put it on ice for 1min. (These procedures must be carried out in the ultra-clean bench.)
d. Set parameters of the Bio-Rad® electroporation system: C = 25 μF, PC = 200 ohm, V = 2.5 kv
e. Transfer the cells to a chilled 2-mm wide electroporation cuvette.
f. Quickly place the cuvette into the electroporation tank for electroporation
g. Take out the cuvette and insert it into ice quickly for standing after the electroporation
h. 5 min later, transfer the cells immediately into 500 μl of TY liquid medium without antibiotics and incubate for 2-3 h at 28°C with shaking at 150-180 rpm
i. Centrifuge at 66000 rpm for 1 min to collect Agrobacterium cells
j. Aspirate 450 μl of the supernatant and re-suspend the cells
k. Spread 50 μl of the re-suspended cells on a solid TY medium plate containing corresponding antibiotics (uneven method on both sides)
l. Place the plates upside down in incubator at 28℃ for 2-3 days.
m. The positive transformants are verified by PCR of the bacterial solution and cultured with streak plate method
a. Prepare 10% NaClO solution.
b. Pick carrot leaves with green colour, healthy condition and suitable size.
c. On the ultra-clean Workbench, pour NaClO solution into a container with carrot leaves, shake it for 8 minutes to make sure the leaves are completely sterilized
d. Pour out the NaClO solution carefully
e. Wash away the residual solution on the leaves with sterile water for 2 or 3 times
f. Absorb the water left on the leaves with dry, sterilized filter paper
g. Transfer the sterilized carrot leaves on MS preculture medium plates in dark at 25 °C for 3 days
a. On the ultra-clean Workbench, cut the petiols of precultured carrot leaves into small pieces using a sharp knife dropped with infection solutions
b. Co-cultured the Agrobacterium infected petiol pieces on B5 media containing 50 µM acetosyringone in dark at 25 °C for 3 days
c. Transferred the co-cultured petiol pieces on MS medium supplemented with 500 mg/ L cefotaxime (Sigma) in dark at 25 °C to induce hairy roots
d. Mark successful transformants with the help of a fluorescence microscope
e. Remove the positive hairy roots from petiol pieces and transfer them on MS plates (without cefotaxime) in dark at 25 °C
a. On the ultra-clean Workbench, cut the petiols of precultured carrot leaves into small pieces using a sharp knife dropped with infection solutions
b. Select the hairy-root tips in good condition and transfer them into Ms liquid medium with Cefotaxime at a final concentration of 200 mg/L
a. Incubate the hairy-root tips in a shaker protected from light at 25oC and 90 rpm with weekly changes of medium.
b. After about 30 days of incubation, add β-estradiol solution and make sure it reaches a final concentration of 2 μM
c. Continue to culture the hairy roots for 120 h in a shaker for anthocyanin synthesis.
a. a)After 120 h of anthocyanin synthesis induction, remove hairy roots from Ms liquid medium wash them with sterile water for 3 - 5 times.
b. Snap-frozen hairy roots with liquid nitrogen and ground them into powder.
c. Freeze-dried the hairy root powder for about 48 h
d. Weigh 10 mg of dried root powder and transfer it into a 2 ml eppendorf tube
e. Add 1 mL of acidified 80% methanol solution (containing 2% formic acid) into the tube and mix well by vortex shaking
f. Incubate the mixture at 60 °C for 15 min.
g. Remove the mixture on ice and then keep it in fridge at 4 °C overnight
h. Centrifuge at 2500 rpm for 10 min and remove the supernatant to new tubes. (The supernatant is anthocyanin extract.)
The total anthocyanin content of the samples is calculated based on the standard curve of the absorbance of cyanidin 3-O-rutinoside (C3R) at 530 nm and 657 nm in the concentration range of 0.001 - 0.05 mg/mL and the result is measured in “relative unites anthocyanins / g dry weight”. The calculational formula of the relative contents of anthocyanins (RCA) is:
The colour changes of anthocyanins in different environments are tested by configuring phosphate buffers of different pH from 4 to 7.