Auxo-Agro™ LBA4404 Chemically Competent Cells

GoldBio’s new Auxo-Agro™ competent cells are methionine auxotrophic strains of Agrobacterium which reduce overgrowth during the infection process while increasing plant transformation efficiency.

Our LBA4404 strain of Agrobacterium tumefaciens can be used in genetic transformation of tomato, tobacco and other plants. After transformation, antibiotics are commonly used to remove Agrobacterium. However, even in the presence of antibiotics, there can be overgrowth of the Agrobacterium strain which can create a more difficult experimental protocol. Auxo-Agro™ cells help to solve this problem when selection is performed in both minimal media without Methionine in combination with selective antibiotics, such as Timentin, Cefotaxime or Meropenem.

GoldBio’s LBA4404 Agrobacterium chemically competent cells allow you to obtain high transformation efficiency in applications such as gDNA or cDNA library construction. Our LBA4404 strain harbors a rifampicin resistance (rif) gene. Furthermore, LBA4404 has an octopine-type Ti plasmid pAL4404 without self-transport function, containing the vir genes.

These strains represent the only auxotrophic strains of Agrobacterium available in the public domain.

Recent literature shows that knocking out genes to cause auxotrophy DOES NOT affect transformation capacity.¹

GoldBio’s LBA4404 Agrobacterium strain was generated, and primary clone supplied by Dr. Wayne Parrott under license from his institution.

Prías-Blanco M, Chappell TM, Freed EF, Illa-Berenguer E, Eckert CA, Parrott WA. An Agrobacterium strain auxotrophic for methionine is useful for switchgrass transformation. Transgenic Res. 202231: 661-676. doi: 10.1007/s11248-022-00328-4. PMID: 36239844. Important note from this paper: “Switchgrass transformation was chosen to validate these strains by evaluating their performance in a difficult transformation system.”

Kit Components

• Competent Cells
• 1 x 12 mL Recovery Media
• 1 x 25 µL Control Plasmid (pCAMBIA1391z Control, 10 ng/µL)

Product Specifications
Competent cell type: Chemical Competent
Species: A. tumefaciens
Strain: LBA4404∆Met
Format: Tubes
Transformation efficiency: ≥1 x 10⁴ cfu/µg pCAMBIA1391z DNA
Blue/white screening: No

Storage/Handling: This product may be shipped on dry ice. LBA4404 Agrobacterium Chemically competent cells should be stored at -80°C, pCAMBIA1391z Control DNA should be stored at -20°C and recovery medium should be stored at 4°C immediately upon arrival. When stored under the recommended conditions and handled correctly, these products should be stable for at least 1 year from the date of receipt.

Reagents Needed for One Reaction

• LBA4404∆Met Chemically Competent Agrobacterium: 50 µL
• DNA (pCAMBIA1391z Control, 10 ng/µL): 5 µL
• Recovery medium: 12 mL

Table 1: Antibiotic disc sensitivity for GoldBio’s Agrobacterium strains (using standard BD antibiotic discs)

Quality Control

Transformation efficiency is tested by using the pCAMBIA1391z control DNA supplied with the kit and using the protocol given below. Transformation efficiency should be ≥1 x 10⁴ CFU/µg pCAMBIA1391z DNA. Our untransformed cells are tested for appropriate antibiotic sensitivity.

General Guidelines

• 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.

Note: A high-voltage electroporation apparatus capable of generating field strengths of 16 kV/cm is required.

Calculation of Transformation Efficiency

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

• TE = Colonies/µg/Dilution
  • Colonies = the number of colonies counted
  • µg = amount of DNA transformed in µg
  • Dilution = total dilution of the DNA before plating

Example: Transform 1 µl of (10 pg/µl) control plasmid into 25 µl of cells, add 975 µl of Recovery Medium. Dilute 10 µl of this in 990 µl of Recovery Medium and plate 50 µl. Count the colonies on the plate the next day. If you count 250 colonies, the TE is calculated as follows:

Colonies = 250

µg of DNA = 0.00001

Dilution = 10/1000 x 50/1000 = 0.0005

TE = 250/0.00001/0.0005 = 5.0 × 10¹⁰

Example: Transform 1 µl of (10 pg/µl) control plasmid into 25 µl of cells, add 975 µl of Recovery Medium. Dilute 10 µl of this in 990 µl of Recovery Medium and plate 50 µl. Count the colonies on the plate the next day. If you count 250 colonies, the TE is calculated as follows:

Colonies = 250
µg of DNA = 0.00001
Dilution = 10/1000 x 50/1000 = 0.0005
TE = 250/0.00001/0.0005 = 5.0 × 10¹⁰

Licensed Product Terms of Sale

1. THIS MATERIAL IS FOR RESEARCH USE ONLY AND NOT FOR USE IN HUMAN SUBJECTS.
2. Materials are understood by buyer to be experimental in nature and may have hazardous properties. Unless prohibited by law, buyer assumes all liability for claims for damages against it by third parties that relate to or arise from the use, storage, or disposal of the purchased materials.
3. Buyer agrees to use the purchased materials in full compliance with applicable law and regulations.