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°C. Store as glycerol stock (800 ml of fresh overnight culture + 200 ul sterile 80% glycerol) at ¬80°C.
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°C 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°C 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°C 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°C.
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°C.
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 ul of LB (or 2YT).
8. Plate all of the suspension on appropriate antibiotic-LB (or 2YT) plates and incubate for two days @ 30°C. 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°C 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°C 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°C 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°C. 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 100ul 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°C
Electroporation
1. Remove tube of competent cells from freezer and place on ice. Allow to thaw slowly on ice.
2. Aliquot 50 ul into each of two 0.5 ml microfuge tubes. Add 1-2 ul 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 ul on selective media (i.e. antibiotic selection for both the bacterial host strain and the plasmid)
9. Incubate plates 2 days at 28-30°C, 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.
According to the strain of Agrobacterium used, different antibiotics are required. The most commonly used strains LBA4404 and GV3101 carry chromosomal resistance to rifampicin, each has a Ti-plasmid (carrying vir [virulence] genes) which encodes another antibiotic resistance. This is summarised in the table below. GV3101 are the same as ‘C58C1RifR-pMP90'. When dealing with a C58-derived strain and tetracycline selection, it is worth doing a control transformation to see what rate of 'spontaneous' resistance to the antibiotic develops, since the appearance of many colonies my not necessarily mean a successful transformation. In fact, I would advise you not to use tetracycline with any Agrobacterium strain which you uncertain about with regards to spontaneous resistance to tetracycline - assume the strain will become resistant until you prove otherwise. With the pGreen system, co-transform with pSoup, and select on kanamycin.
Strain Chromosomal selection Ti-plasmid Selection
LBA4404 Rifampicin pAL4404 Streptomycin
GV3101 Rifampicin pMP90 Gentamicin
Generally rifampicin and kanamycin can be used at 50ug/mL, streptomycin at 30ug/mL, gentamicin at 25ug/mL, tetracycline at 10ug/mL. Tetracycline resistance encoded by pSOUP, however, should be selected at 2ug/mL (‘lo-Tet’)
General-purpose chemical transformation of Agrobacterium
Click here to PRINT this protocol
****Tubes of competent Agrobacteria made using this method can be frozen and -80 degrees Celsius and re-used at a later date - see notes****
This method works very well with the pBin19-derived and pGreen plasmids encoding bacterial kanamycin resistance. Chemical co-transformation: Around 500 colonies are obtained per tube with 10 micrograms of pSOUP, and around 20 colonies per tube co-transforming with both pSOUP and pGreen. This method was optimised to deal with the transformation of LBA4404 with pSOUP, a weak tetracycline resistance conferring plasmid.
If you are working with pGreen, you might also like to visit the pGreen bulletin board.
According to the strain of Agrobacterium used, different antibiotics are required. The most commonly used strains LBA4404 and GV3101 carry chromosomal resistance to rifampicin, each has a Ti-plasmid (carrying vir [virulence] genes) which encodes another antibiotic resistance. This is summarised in the table below. GV3101 are the same as ‘C58C1RifR-pMP90'. When dealing with a C58-derived strain and tetracycline selection, it is worth doing a control transformation to see what rate of 'spontaneous' resistance to the antibiotic develops, since the appearance of many colonies my not necessarily mean a successful transformation. In fact, I would advise you not to use tetracycline with any Agrobacterium strain which you uncertain about with regards to spontaneous resistance to tetracycline - assume the strain will become resistant until you prove otherwise. With the pGreen system, co-transform with pSoup, and select on kanamycin.
Strain Chromosomal selection Ti-plasmid Selection
LBA4404 Rifampicin pAL4404 Streptomycin
GV3101 Rifampicin pMP90 Gentamicin
Generally rifampicin and kanamycin can be used at 50ug/mL, streptomycin at 30ug/mL, gentamicin at 25ug/mL, tetracycline at 10ug/mL. Tetracycline resistance encoded by pSOUP, however, should be selected at 2ug/mL (‘lo-Tet’)
Chemicals & Solutions
Use LB supplemented with magnesium sulphate.
The magnesium sulphate appears to reduce clumping in LBA4404 which can hamper transformation as well as make resuspension difficult, this isn't important for GV3101, so the magnesium can be left out. I don't bother to pH the LB, because I checked that the first time I made it with a certain set of reagents and it was indeed about pH 7.5
TYNG =10g/L bacto-tryptone
5g/L yeast extract (YE)
5g/L NaCl
0.2g/L MgSO4
pH 7.5
Transformation solution: 20mM CaCl2 (ice-cold)
Equipment
Liquid nitrogen or dry ice
Procedures
DAY1
Grow Agrobacterium LBA4404 pAL4404 with selection overnight at 27-28 degrees celsius in 5mL TYNG medium
DAY2
Early the next day (DAY2), vortex to break up clumps (be brutal!)
On or after about 4pm the next day (DAY2), inoculate 60mL TYNG (with same selection) with 0.5mL of starter culture
Incubate at 28°C with 225-250rpm shaking overnight
DAY3
Cool centrifuge, cool tubes and sterile CaCl2 on ice
Around midday put culture on ice for 10 mins
Spin down bugs 4800rpm for 6 mins at 4°C, get rid of supernatent
Rinse with 1mL 20mM CaCl2 (ice-cold) and spin briefly again (gets rid of excess antibiotics which would might kill the Agro during the recovery period)
Resuspend in 1mL 20mM CaCl2
Take 150uL of Agro cells on ice, and add to upto 10 microlitres of pSOUP (upto 10ug)
Flick to mix and freeze in liquid nitrogen for 5 minutes
Remove tube and thaw, takes about 5-10 minutes on the bench top in our lab
Add thawed cells to 1mL LB in 20mL sterilin tube, incubate with shaking (200rpm) overnight.
Aliquot and freeze remainder of cells, storing at -80°C, to be used with reasonable transformation efficiency at a later date.
DAY4
Pour plates TYNG with selection (Rif plus antibiotics whose resistance is conferred by transforming plasmid)
Again, I reiterate: DO NOT USE TETRACYCLINE AS A SELECTABLE MARKER unless you are sure your strain will not become spontaneously resistant!
16 hours into the recovery period, plate onto TYNG. The transformation can be incubated on the bench top so long as the ambient temperature does not exceed 30°C, but are best grown at 28°C in an incubator [You can cut the recovery period to a few hours, with good results]
DAY 5-6 Too early to see bugs
DAY 7 Should see very small colonies growing
DAY 8 Colonies visible
Note 1: In an example where pSoup (a tetR) plasmid was transformed into LBA4404, there is no selection for the Ti plasmid at the end, so bugs should be streaked onto media containing Strep as well as Rif and Tet (without pGreen), or if a co-transformation has been done Strep and Kan as well as Rif and Tet (pSOUP + pGreen maintenance). In this case, I am sure LBA4404 do not become spontaneously resistant to tetracycline. If I had been using a GV strain, I would have had to select only for the pGreen using Kan and would on no account have used Tet!!!
Note 2: The 'half-life' of pGreen without kanamycin is 1 day, so do not leave on plates for too long! Regularly restreak!
Note 3: Actually LB works very well if you feel to lazy to make up TYNG which is really sweet LB, but Mg does help stop clumping.
Note 4: Re-use of competent Agrobacteria - remove frozen cells from freezer, pipette a few microlitres of transforming plasmid directly onto frozen cells, thaw using warmth of hand, flick to mix several times (5-10 secs), close tube and drop directly into liquid nitrogen. Allow tube to incubate in liquid nitrogen for 5 minutes and continue with protocol as before.
To be sure the insert in the binary vector is intact, do a plasmid mini-prep on the Agrobacterium clone to be used for plant transformation, then transform it back into E.coli. A mini-prep of plasmid from this can be used for confirmation of the intactness of the insert, sequenced, and proves the kanamycin-resistant trait is still held on an easily transferable and therefore episomal entity, rather than having integrated into the Agro chromosome which would also give you a kanamycin resistant bug.
MOLBIOL.NET - Premier Portal for Bioinformatics and Molecular Biology Tools
