Intact Genomics (ig^®) K599 Chemically Competent Agrobacterium cells are made from a specific strain of  Agrobacterium rhizogenes, Agrobacterium rhizogenes (str R) pRi2659 (agropine type). K599 was originally isolated from cucumber exhibiting hairy root disease symptoms and has been widely used for hairy root transformation(1).

Agrobacterium rhizogenes is a soil-borne gram-negative bacterium that can infect most dicotyledons, a few monocotyledons and some gymnosperms.  K599 Chemically Competent Agrobacterium are optimized for the highest transformation efficiencies and are useful for transgenic operations of corn, soybean (wild soybean), cotton, peanut, dandelion, cowpea and other plants.  K599 Agrobacterium rhizogenes strain contains pRi2659 agrobacterium-type Ri plasmid and displays streptomycin resistance.

SpecificationsCompetent cell type: Chemically CompetentSpecies: A. rhizogenesStrain: K599Format: TubesTransformation efficiency:  ≥ 1 x 10⁴  cfu/µg pIG7spe DNABlue/white screening: NoShipping condition: Dry ice

Reagents Included

• K599 Chemically Competent Agrobacterium
• DNA (pCAMBIA1391z, 500 pg/µl)
• Recovery medium

Note: Liquid nitrogen is required.

StorageK599 Chemically Competent Agrobacterium: -80 ºCpCAMBIA1391z control DNA: -20 ºCRecovery medium: 4 ºC

Quality ControlTransformation efficiency is tested by using the pCAMBIA1391z control DNA supplied with the kit and using the protocol in this manual. Transformation efficiency should be ≥1 x 10⁴ CFU/µg pCAMBIA1391z. Untransformed cells are tested for appropriate antibiotic sensitivity.

General Guidelines

Follow these guidelines when using K599 Chemically Competent Agrobacterium cells:

• Handle competent cells gently as they are highly sensitive to changes in temperature or mechanical lysis caused by pipetting.
• Thaw competent cells on ice, and transform cells immediately following thawing. After adding DNA, mix by tapping the tube gently. Do not mix cells by pipetting or vortexing.

Example Calculation of Transformation EfficiencyTransformation Efficiency (TE) is defined as the number of colony forming units (cfu) produced by transforming 1µg of plasmid into a given volume of competent cells.

TE = Colonies/µg/Plated

Transform 1 µl of (500 pg/µl) pCAMBIA1391z control plasmid into 50 µl of cells, add 950 µl of Recovery Medium. Recover for 3 hours and plate 100 µl. Count the colonies on the plate in two days. If you count 5 colonies, the TE is calculated as follows:

Colonies = 5µg of DNA = 0.0005Dilution = 100/1000 = 0.1TE = 5/.0005/.1 = 1×10⁵

Please note, all agrobacterial strains are not well studied for antibiotic resistance and there are many agrobacterial strains.  Therefore, it is the customer’s responsibility to make sure his/her vectors are compatible with the Agrobacterial strains if he/she uses an alternate antibiotic selection than spectinomycin-selection.

References

1. Combard, A.; Brevet, J.; Borowski, D.; Cam, K.; Tempé, J. Physical map of the T-DNA region of Agrobacterium rhizogenes strain NCPPB2659. Plasmid 1987, 18, 70–75.

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