A4 Agrobacterium Strain Information

Genotype

Agrobacterium rhizogenes (kan^R) Ar A4 Ri (agropine type)

Technical Parameters

Pricing & Ordering

A4 Agrobacterium Strain Description

A4 Agrobacterium tumefaciens is a natural Agrobacterium alkaline strain. It has kanamycin resistance and can be cultured on TY medium. The optimal temperature for culture is 30°C. Agrobacterium rhizogenes is a Gram-negative soil bacterium belonging to the genus Agrobacterium in the Rhizobiaceae family. It can infect most dicotyledonous plants, a few monocotyledonous plants, and a few gymnosperms. The Ar.A4 Agrobacterium tumefaciens strain contains the Agrobacterium tumefaciens Ri plasmid, which has a wide range of hosts (corn, tobacco, carrots, licorice, etc.) and is resistant to kanamycin.

Agrobacterium rhizogenes is a Gram-negative soil bacterium belonging to the genus Agrobacterium in the family Rhizobiaceae. It can infect most dicotyledonous plants, a few monocotyledonous plants, and certain gymnosperms. The Ar.A4 strain of Agrobacterium rhizogenes contains an Ri-type Ti plasmid, exhibiting a broad host range (such as corn, tobacco, carrots, licorice, etc.) and kanamycin resistance. This product is a second-generation glycerol stock (propagated for two generations from the original strain), with no extensive amplification. The nuclear DNA and phenotypic stability are maintained, and the colony phenotype and growth curve match the original strain with over 99.5% accuracy.

Operating Instructions

1. Upon receiving the strain, customers must not directly inoculate and propagate the bacterial liquid. The strain should first be revitalized by allowing single colonies to grow before picking and expanding them. The revitalization method is as follows: Open the lid under a biosafety cabinet, use a loop to pick up a small amount of glycerol bacterial liquid, and gently streak the TY plate surface using the streak plate method (Note: Avoid piercing the medium). Seal the plate and incubate at 30°C for 3-5 days to allow single colonies to grow.
2. Cross-streaking method: The purpose of streaking is to achieve a gradient dilution effect through several discontinuous streaks, ensuring the growth of single colonies. The specific steps are as follows:
   1. The glycerol stocks retrieved at -80°C should have their lids opened in a biosafety cabinet, and the bacterial suspension/colonies should be streaked immediately without waiting for thawing. Solid-state samples can be picked up with an inoculating loop for streaking. The key operation principle is "rapid handling and immediate streaking" to minimize temperature fluctuations' "cold shock" and physical damage from ice crystals. If receiving liquid glycerol stocks, streaking can be performed directly by opening the lid in a biosafety cabinet and using an inoculating loop to pick up a small amount of bacterial suspension for streaking.
   2. Remove the sterile inoculation loop, ensuring no contamination. Divide the plate into five zones. Three straight lines are drawn in each zone. The first and second zones can be streaked with the same loop. After streaking zones 1 and 2, replace the loop with a new one for zone 3, then another new loop for zone 4, and yet another for zone 5. Each time the loop is changed, the streaking must pass over the previous streak marks, effectively diluting the bacterial culture. A simplified method may involve streaking only three or four zones.
   3. Customers can also use a metal inoculating loop, sterilize it by flaming, and streak it.

Antibiotics for Agrobacterium

LB and YEB Media Formulations

TY Formula (1 L)

• Tryptone 5 g
• Yeast extract 3 g
• Add water to 1 L, dissolve completely, autoclave at 121°C for 20 min
• Prepare 1 M calcium chloride aqueous solution, autoclave at 121°C for 20 min
• Add 10 mL sterile 1 M calcium chloride solution per 1 L sterilized TY liquid medium

* For TY solid medium, add 15 g agar powder.

Electroporation of Agrobacterium tumefaciens

1.1 Preparation of Electrocompetent Cells

See Lin (1995) for additional information.

1. Inoculate 1.5 L of YM broth in a 2.8 L Fernbach flask with an aliquot from log phase culture of A. tumefaciens.
2. Incubate at 30°C overnight, shaking at 300 rpm to a density of 5-10x 107 cells/mL.
3. Decant the cells into sterile 500 mL centrifuge bottles and pellet the cells by centrifugation at 3,000 × g for 10 min at 4°C.
4. Carefully pour off and discard the supernatant; place the centrifuge bottles with the cell pellets on ice.
5. Add ~50 mL of sterile, ice-cold 10% glycerol to each bottle and vortex to resuspend the cell pellets; bring the volume in each centrifuge bottle to 500 mL with sterile, ice-cold 10% glycerol. Pellet the cells by centrifugation at 3,000 × g for 10 min at 4°C; pour off and discard the supernatant.
6. Wash the cells again as in step 5.
7. Resuspend each cell pellet in 5 mL of sterile, ice-cold 10% glycerol and transfer to a chilled 30 mL Oakridge tube. Pellet the cells by centrifugation at 3,000 × g for 5 min at 4°C; pour off and discard the supernatant.
8. Resuspend the cell pellet in 0.5 mL of sterile, ice-cold 1 M sorbitol; the final cell volume should be ~1.5 mL and the cell concentration should be ~5 × 10¹cells/mL. Dispense 200 μL aliquots of the electrocompetent cells into sterile 1.5 mL microfuge tubes; freeze the cells in an isopropanol-dry ice bath, then store at -70°C. The cells are stable for about 6 months under these conditions.

1.2 Electroporation

1. Pipette the DNA samples (up to 5 μL) to be electroporated into sterile 1.5 mL microfuge tubes; the DNA should be in either water or TE. Place tubes on ice.
2. For each DNA sample to be electroporated, add 1 mL of YM broth to a 17 × 100 mm tube at room temperature, and place a 0.1 cm electroporation cuvette on ice.
3. Thaw the electrocompetent A. tumefaciens cells on ice. For each DNA sample to be electroporated, add 20 μL of electrocompetent cells to each DNA sample; gently tap the tubes to mix.
4. Set the MicroPulser to "Agr".
5. Transfer the DNA-cell samples to the electroporation cuvettes and tap the suspension to the bottom of the tube. Place the cuvette in the chamber slide. Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber. Pulse once.
6. Remove the cuvette from the chamber and immediately use the YM broth in the 17 × 100 mm tube to transfer the cells from the cuvette to the tube.
7. Check and record the pulse parameters. The time constant should be about 5 milliseconds. The field strength can be calculated as actual volts (kV) / cuvette gap (cm).
8. Incubate the cells 3 hr at 30°C, shaking at 250 rpm. Plate aliquots of the electroporated cells on YM agar plates containing the appropriate selective media. Incubate plates for 48 hrs at 30°C.

1.3 Solutions and Reagents for Electroporation

YM Broth (1 L): 0.4 g yeast extract, 10 g mannitol, 0.1 g NaCl, 0.1 g MgSO4 0.5 g K₂HPO₄.H₂O. Dissolve in 1.0 L water and adjust to pH 7.0. Autoclave. For YM plates, add 15 g agar per 1 L of YM broth.

Frequently Asked Questions