Agrobacterium growth and transformation

Growth and storage of Agrobacterium tumefaciens

Strain GV3101: resistant to gentamycin and rifampicin so add 25-50 ug/ml Gentamycin, 10 ug/ ml rifampicin on plates or in liquid media for selection. GV3101 is sensitive to kanamycin (or chloramphenicol), so is a good strain for use with binary vectors that confer kan resistance (or chloramphenicol resistance) in bacteria (e.g. transformed cells will be Gent, rif and Kan resistant). GV3101 carries a disarmed Ti plasmid that possesses the vir genes needed for T-DNA transfer, but has no functional T-DNA region of its own. Grow at 28-30℃. Store as glycerol stock (800 ml of fresh overnight culture + 200 μl sterile 80% glycerol) at  -80℃.

Transformation of A. tumefaciens with plasmid DNA (binary vector system)

Two methods of direct DNA transfer can be used, thus eliminating the need for the old genetic method of transferring a plasmid maintained in E. coli by triparental mating (E. coli + RK2013 + A.tumefaciens).

Method 1: Freeze/thaw shock transformation. This is not very efficient (~200 colonies per microgram of DNA), but it is really easy.

1. Pick a single colony of the Agrobacterium strain of choice and inoculate 3 ml of LB (or 2YT) in a 15 ml snap-cap tube (Falcon tube). Grow @ 30℃ overnight on a roller drum. Be sure to include the appropriate antibiotic selection (Gentamycin, rifampicin for GV3101).

2. Inoculate 50 ml of LB in a 250 ml flask with 0.5 ml (1/100 volume) of the overnight culture and grow @ 30℃ until mid-log (OD₆₀₀ is between 0.5 and 1.0). In practice, I (Craig) never bother taking OD readings but grow cultures until they are dense enough to give nice, silky, cloud-like swirls of cells when the culture is held up to the light and jostled. This takes ~4-5 hours to get the cells to this stage. You could probably cut down the time by increasing the initial inoculum.

3. Chill culture 5-10 min. on ice, centrifuge @ 3000 rpm for 5 min. @ 4℃ in chilled, sterile centrifuge tubes (30 ml Corex tubes).

4. Discard supernatant, drain inverted for 30-60 seconds, and resuspend pellet in 1 ml of ice cold 20 mM CaCl₂. Dispense 0.1 ml of bacterial suspension into each of two pre-chilled 1.5 ml. microfuge tubes on ice. One is a control.

5. Add 1 ug of plasmid DNA to one tube and nothing to the other the control) and mix by tapping with your index finger. Freeze tubes in liquid N2, then thaw tubes for 5 min. @ 37℃.

6. Add 1 ml of LB (or 2YT) to each tube, transfer content to a 15 ml snap-cap tube and incubate for ~2 hours on a roller drum @ 30℃.

7. Pour contets into a 1.5 ml microfuge tube and spin tubes 5 minutes at ~4K rpm to pellet cells. Remove supernatant and resuspend pellet in 100 μl of LB (or 2YT).

8. Plate all of the suspension on appropriate antibiotic-LB (or 2YT) plates and incubate for two days @ 30℃. Transformed colonies should be visible on the second day of incubation.

Method 2: Electroporation

This method is much more efficient and one can make a batch of frozen competent cells and store them at ¬80℃ for multiple experiments, which is convenient if you plan to be doing lots of cloning and transformations.

Preparation of competent cells

1. Pick a single colony of the Agrobacterium strain of choice and inoculate 3 ml of LB (or 2YT) in a 15 ml snap-cap tube (Falcon tube). Grow @ 30℃ overnight on a roller drum. Be sure to include the appropriate antibiotic selection (e.g. Gentamycin for GV3101).

2. Inoculate two 500 ml flasks each containing 100 ml of LB with 0.5 ml (1/100 volume) of the overnight culture and grow @ 30℃ with vigorous shaking until mid-log (OD600 of 0.5 - 1.0). In practice, I (Craig) never bother taking OD readings but grow cultures until they are dense enough to give nice, silky, cloud-like swirls of cells when the culture is held up to the light and jostled. It takes ~4-5 hours to get the cells to this stage. You could probably cut down the time by increasing the initial inoculum.

3. Fill six 30 ml Corex tubes with the culture and spin 5 min at 4K in a JA-20 rotor at 4℃. Pour off supernatant and drain tubes inverted for ~60 seconds.

4. Resuspend cells in each tube with 12-15 ml (~1/2 volume) ice-cold 10% glycerol. Repeat spin.

5. Resuspend cells in each tube in 4 ml of ice-cold 10% glycerol. Combine and aliquot into 2 tubes. Repeat spin.

6. Resuspend cells in each of the two tubes in 2ml of ice-cold 10% glycerol. Combine into one tube. Repeat spin (use a balance tube with water or 10% glycerol).

7. Resuspend final pellet in 1.5 ml ice-cold 10% glycerol.

8. Dispense 100μl aliquots into fifteen 1.5 ml microfuge tubes pre-chilled on ice. Each tube will have enough cells for 2 transformations. Quick-freeze the tubes in liquid nitrogen and store at  -80℃

Electroporation

1. Remove tube of competent cells from freezer and place on ice. Allow to thaw slowly on ice.

2. Aliquot 50 μl into each of two 0.5 ml microfuge tubes. Add 1-2 μl of DNA (10-50 ng is plenty; DNA can be in TE buffer or water) and mix by tapping the tubes.

3. Transfer cells + DNA to pre-chilled (on ice) electroporation cuvettes with either1 or 2 mm gap sizes. Make sure the white cuvette holder from the Bio-Rad machine is also pre-chilled on ice.

4. Take the ice bucket with the cuvettes and cuvette holder to the gene pulser. For cuvettes with a 2mm gap size, adjust the Gene Pulser unit "Set Volts"setting to 2.5 kV and the "CAP" setting to 25 uFD. Set the resistance to 400 Ohm on the Pulse Controller Unit (note that this is higher than for E. coli, which is usually set for ~200 Ohm).

5. Place the cuvette in the cuvette holder, slide down to engage the electrodes and push both buttons on the Gene Pulser, holding them until the tone sounds. Push the time constant display and note the time which should be ~9 msec (optimum conditions are reported to be those that give time constant between 8-12msec).

6. Add 1 ml of growth medium directly to the cuvette immediately after the pulse (i.e. before doing the next sample) and place on ice.

7. Transfer contents of cuvette to 15 ml Falcon tube and incubate on roller drum at 30C for ~2 hours.

8. Plate 100-200 μl on selective media (i.e. antibiotic selection for both the bacterial host strain and the plasmid)

9. Incubate plates 2 days at 28-30℃, at which time colonies should be visible.

The first time I did this procedure myself, I used 250 ng of DNA and had thousands of colonies on the plate (and this was after plating only 1/10 of the transformed cells). In contrast, the freeze/thaw method gave a total of several hundred colonies using 4-fold more DNA and 10-fold more cells. Therefore, electroporation using frozen competent cells is 102-103 more efficient than the freeze/thaw method.