Hello everyone,
I am having some trouble in transforming Agrobacterium cells with the Gateway vector pAMPAT.
For the time being, I have only tried GV3101 and have not managed to make it work yet.
This is what I have been doing:
Preparation of Agrobacterium electrocompetent cells:
- Grow Agrobacterium from a 25% glycerol stock on LBA + Rifampicin, Gentamicin for 2-3 days at 28oC
- Pick a single colony and grow Agrobacterium in a liquid culture (5 ml - same antibiotics and as above) / incubation at 28oC, 250 rpm shaking
- Inoculate 100ml of LB with 5 ml of the liquid culture and incubate for approx. 6-8h, until OD500 0.5
- Transfer into 50 ml Falcon tubes and:
- Centrifuge at 4000 rpm, 4oC for 15 mins
- Resuspend pellets in 25ml of cold sterile dd-water
- Repeat the above 2 steps and add 25 ml of cold 10% sterile glycerol instead of dd-water
- Repeat and add 400μl of cold 10% sterile glycerol
- Make 50μl aliquots, freeze in liquid nitrogen and store at -80oC
Electroporation:
- Add 800ng of plasmid (pAMPAT expressing the gene of interest with a YFP tag) in 50μl Agrobacterium electrocompetent cells
- Gently homogenise and transfer into 1.0 mm cuvettes
- Electroporation at 1440V, using the Eppendorf electroporator 2510
- Add 500μl of SOC medium right after the electroporation
- Plate cells on LBA + selective antibiotics (carbenicillin 50 - for pAMPAT, gentamicin 20 - for Agrobacterium)
- Incubate for 2-3 days at 28oC
Until today, I have not seen any colonies (after 3-4 days).
What I am going to do next is use a different strain.
Has anyone transformed Agrobacterium - GV3101 or any other strain - with pAMPAT or any other gateway vector?
If yes, which method/ steps/ conditions did you use?
Any suggestions/advice would be much appreciated.
Thanks very much in advance.
Best wishes,
Nikolaos (Nikos) Mastrodimos
Can the home-made Agrobacterium electrocompetent cells grow on plates (without antibiotic) and on plates (with Gentamicin)?
Nikolaos Mastrodimos
Have you tried *Ampicillin* (see attachment; click to see the entire document), instead of Carbenicillin? https://www.ncbi.nlm.nih.gov/nuccore/AY436765
Yuan-Yeu Yau thank you for your response. I have not checked that, but it sounds like a good idea. Are you suggesting that the cells might die while making them electrocompetent?
About ampicillin, no, I have not tried that either, as I have never had issues with carbenicillin (with pAMPAT or other vectors). Have you ever had ampicillin working and carbenicillin not? Many thanks again
Nikolaos Mastrodimos
1. It is better you check it. Anything could happen doing this kind of experiment (from my personal experience).
2. No, I have not used carbenicillin to select for binary vector (in Agrobacterium cells). I used mostly kanamycin-resistant binary vectors. I only use carbenicillin to eliminate Agrobacterium during transformation.
Yuan-Yeu Yau, thanks so much for your insight.
May I ask you if the binary vectors you have used for transforming Agrobacterium are Gateway vectors? If yes, could you please tell me which one(s)?
Nikolaos Mastrodimos
No, I have not done plant transformation with Gateway vectors. I use pBI121 and pCambia series. One thing, I heard of that some Gateway vectors can be lethal to Agrobacterium. Have you noticed this and took prevention? (see attachment, [1])
(1) A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens (nih.gov)
Yuan-Yeu Yau , I am doing the screening for the expression vectors (pAMPAT: gene of interest) in E. coli, so I do not need to vectors with ccdB for negative selection in Agrobacterium.
I guess there is a possibility that pAMPAT is lethal to Agrobacterium indeed, hence, this might be the issue here.
I think will try another Gateway destination vector and see how it goes.
Many thanks again for your answers.
Nikolaos Mastrodimos
You are welcome, and let me know your new results of using another Gateway destination vector, to see if it works. Good to talk to you, and good luck.
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