Hi,

This might be due to large numbers of cells and less effective antibiotic. You may please check your antibiotic stock..it it is old replace it....this may be one of the reason

Hi

Thanks for the reply but I made new stocks for antibiotics and made new plates with that.The problem is still there.

Hi Sony,

The  other factors for this reason cane be:

1)  if the plasmid amount used for transformation in very high and if the number of cells are also high. You can dilute the cells and plate the cells.

2) Also if the competent cell preparation is contaminated with some other resistant strain...it will out grow the recombinant strain and will result into a lawn.....in this case dilution may not be of much help...you'll have to prepare the cells again..

bye

Ok let me explain what I did till now,I have changed the plasmid conc. from 10ng-800ng.I have plated different dilutions of bacterial culture.I prepared fresh competent cells and used them.I did almost all the troubleshooting from my side.

This may be a dumb question, but are you adding the antibiotics before autoclaving the media or after?

And at which temperature are you growing your agros?

isolate single clones and check by PCR for your plasmid and for Agrobacterium genes. If everything is ok, use the cells for whatever you have designed the vector

Agrobacterium strain GV3101 should be rifamycin resistant (chromosomal mutation) so use rifamycin to select for only GV3101 cells if you think you have contaminating bacterial cells or just to provide selection for the GV3101 cells and for plasmid separately. 

Perhaps it would be best to clone and transform ligation to E. coli first, then characterize the clone, do a plasmid prep of that selected clone then electroporate the plasmid clone into GV3101 and select with rifamycin and the antibiotic selection for plasmid clone (T-DNA vector). 

I would try to use a cloning plasmid vector (T-DNA) that confers kanamycin resistance since kanamycin works well for selection in Agrobacterium.

Hope this helps!

Hi ! 

Most of the solutions are already given above. Next time try one of these things. 

1. Dilute ur transformed agro in 10x, 100x and 1000x and plate them separately on selectable media. Try to choose from the 100x plates. Even if you have never antibiotic higher amount if transformed bacteria cells can use up most of them really quickly and help the non transformed cells to grow. So you would want evenly sparsed cells and not crowded ones (look for the shelf life of these antibiotics at 30 degrees)

2. Make new competent cells , in the worst case scenario. Make your own stocks and don't use them beyond 4 months. Don't autoclave them with media. Filter sterilize them and add them to autoclaved media separately that is cooled to 50 degrees. Use a water bath to maintain the media at 50 until plated. (I believe you are doing this already )

3. You may try to increase the conc. Of the antibiotics in the media a bit if still the problem persists. 

Good luck 

Hi, an other dumb question: Have you performed a negative control by plating on the same selective media non transformed cells?

1. Like others said: the antibiotics expired.

2. try this method, it is easy and worked.

Add about 0,1-1 µg of plasmid to the frozen cells.

Thaw the cells by incubating in a 37°C water bath for 5 min.

Place the cells on ice for 30 min.

Spread the cells on prewarmed LB plates (28°C) containing all the appropriate antibiotics

After 2 days incubation at 28°C about 20-100 colonies will appear.

I totally agree with Michael A Savka. Do the cloning step first in E. coli, and then electroporate the plasmid into Agrobacterium.  Base on our experience, this is relatively easier, and is the tranditional approach in our lab. Good luck.

Agreeing with all the above I would also suggest to you (if you did not do so already) to plate your electro competent Agrobacterium cells to a fresh selective plate without performing the electroporation step to exclude that the Agrobacterium strain you picked is somehow already resistant to your antibiotic of choice. If this is a problem, you might have to look for a vector with another resistance marker to do the transformation (or use another Agrobacterium strain).  Best of luck!

Thanks to all.I would like to tell you again that I am using fresh Competent cells,I have first transformed in E.coli then I am transforming in Agro.Antibiotic I am adding after autoclaving. Negative control is fine.I have used diluted culture for plating but it doesn't worked.

I am following same protocol what Limin Wu has told as one of my friend suggested the same,waiting for result.Thanks again. 

The Agrobacterium grow slow, 12-24 hours is normal, please have patience to wait

If the negative control did not have colonies, the transformed GV3101 could grow on antibiotic plates (including Rifampicin), you can go ahead by streaking the lawn of GV3101 to have single colonies and confirming the exist of transformed plasmid by PCR. Sometimes, GV3101 grows crazy

I followed the protocol what Limin Wu suggested but again I got bacterial lawn after 10 hrs.Most of the suggestions  given by you all have been followed still my problem is existing. 

Plate your bacteria in different plate containing different concentration of antibiotic for e.g std conc to plus and minus 25-50 microgram/ml. Then observe in which concentration you will not get any bacterial colony. select the one minus concentration for your experiments.

Best of luck

Transform the competent cells in E.coli then in Agrobacterium

Add the selective agent (probably rifampicin) by cold sterilization after autoclaving 

Extend the time for colony selection

Try YEB and YEP medium with rif + 0,2 mg/l Phosphinotricin (PPT) for promoting the fast growth of Agrobacterium cells 

Good Luck!!

Hi

Actually my problem is that after transformation I am getting total bacterial lawn not single colony.That is the growth is too much.I am transforming after E.coli transformation. Priti I will do whatever you suggested.

Thanks all. 

Try using AB minimal medium  instead of YEP or LB . Also check whether your Agro is contaminated . on AB minimal medium only Agro grows and with rifampicin you could get single colonies. You could also start your Agro competent cells from single Colo ies and use for transformation.

I think there is some problem with cells competency, if you are using GV3101, many suggestions are here about using rifampicin, i agree with them, secondly, can you tell the dose of rifampicin if you are using and then yow do you sterile.. thirdly, try changing the method of making competent cells.. hope it wil work,,

Rifamycin is prepared in methanol and used at 100 to 150 micrograms per milliliter of media. 

The origin of the resistance is by mutation in the beta-subunit of RNA polymerase that prevents rifamycin from complexing (binding).

Hi

I am using 40 microgram/ml of rifampicin,I don't sterilize it and Ihave made the stock in DMSO.I haven't tried different method to make competent cells.

Which OPINE utilizer is GV3101 and GV3010? Does anyone know the answer? Sorry for the primitive question,

Hi all,

I know this question have been posted a while ago now, but I just want to add something useful for future references regarding the bacterial lawn that can be noticed after just 12 to 24 hours after transformation of Agrobacterium GV3101.

I did notice the same thing now that I am working with Agrobacterium transformation for the GV3101. I am sure to follow every recommendation stated before for my transformation and follow every step in the protocol for transformation (1- Check expiration dates of every antibiotic that you need, 2- Make sure to prepare them correctly, store them correctly and then add them when the media is not hot, 3- Make sure to include a negative control or cells transformed with water or without adding plasmid DNA).

A possible explanation for this observation might be that as part of the Agrobacterium transformation protocol, if you use LB or other liquid media (without antibiotics) to re-suspend your Agrobacterium GV3101 transformed cells after the 2-4 hours recovery stage in LB or other liquid media (again without any antibiotics) and use some micro-liters of the re-suspension to spread your transformed bacteria in the LB or other solid media plates containing your antibiotics, the non-transformed bacteria are able to grow initially in the media that you use for spreading the cells (because of course it does not have any antibiotic on it!), but after a while (24 to 48 hours at 28C, and depending on your antibiotic concentration) the non-resistant bacteria are going to start dying and the lawn is going to disappear from your plate, leaving just resistant colonies (because the antibiotics from the solid media are going to kick in and kill the non-resistant ones but not the resistant ones!). The advantage to always include a negative control is that you can see that the same lawn is going to appear initially, but it wont leave any colony after 24-48 hours when the antibiotics kick in!. Then you would be sure you did not make any mistakes preparing your plates or planning your experiments.

So, my recommendation for other people with this same observation is:

1- Do not throw away your plates if you observe this initial growth in your plates at 12-24 hours at 28C. Even if you can observed them in your negative control (NOTE: just initially!).

2- Be patient and give it time! and you will start seeing the true resistant colonies after the lawn dies.

3- You can also use LB or other liquid media with the appropriate antibiotics for re-suspension (after the required recovery stage!) to spread your cells and avoid the initial lawn observation.

4- Always include a negative transformed control without plasmid DNA on it or non-transformed control to check for possible mistakes. If you observe colonies on the negative controls after the disappearance of the lawn, then your cells are already resistant to the antibiotics and you will need to either change the strain of Agrobacterium, repeat the preparation of your competent cells and of course try the transformation experiment again.

Hope this helps other beginners with this technique.

It is important to carefully consider the antibiotic selection of the plasmid, in this case the T-DNA vector plasmid, and its antibiotic selection (antibiotic resistant gene for bacterial selection). It would be good to test the vir-helper strain for antibiotic sensitivity to the antibiotic that will be used for T-DNA plasmid selection before selecting the vir-helper strain and or T-DNA vector containing its antibiotic resistant gene encoded for bacterial selection. The antibiotic kanamycin or closely related antibiotic gentamycin has been most robust for selection of introduced plasmids in Agrobacteria in my experiences.

Vir-helper strain Agrobacterium GV3101 contains vir-helper plasmid pMP90 (Koncz and Schell 1986, Mol Gen Genet 204:383) that is derived from pTi plasmid pTiC58 - so it contains the nopaline catabolic pathway. The opine, nopaline, can be used as a sole carbon source in screening for the presence of pMP90 and should lead to differential growth on AB minimal media with nopaline as sole carbon source if compared to growth with GV3101 without pMP90 or other bacterial contaminants. The AB minimal media with nopaline as the sole carbon source and supplemented with the antibiotic for the bacterial selectable marker encoding the antibiotic detoxification enzyme on the T-DNA vector plasmid can be used to select GV3101(pMP90) in which your T-DNA plasmid has been introduced. In this case growth should only occur with bacterial cells that contain the nopaline catabolic pathway (encoded by the vir-helper plasmid pMP90) and T-DNA plasmid encoding the detoxification of the antibiotic used to select for your T-DNA vector you are introducing into GV3101 (pMP90) vir-helper strain. Alternatively use Agrobacterium vir-helper strains LBA4404 or EHA105.

Dear Michel, probably these sentences have not been read by you: “obtained with a frequency of 2.1-4.0 x lo-'. Each transcon- jugant was analysed in detail by Southern hybridization and found to contain a new Ti-plasmid, pMP90, from which the pTiC58 KpnI fragments 3, 12 and 13 were absent and which carried an insertion of the Gmr gene in Hind111 fragment 2. The deletion-insertion event had also inacti- vated the nopaline catabolism genes, located to the right of the T-DNA on fragments KpnI(12) and HindIII(2) (Holsters et al. 1980).” So unfortunately zou could not use nopaline as only carbon source. I do not know what is the situation with LH4404 or EHA105. If you hapoopen ti figureout, please, be so kind let me know. Theinformation above came direclz from the Koncz Lab (Max-Planck, Köln, Germany)

INFORMATION FROM CSABA KONCZ TODAY:

1. For selecting for opine catabolism in a minimal media zou should use BTB (bromethzmople-blue)  pH indocator (the opine/cataboliser colobnies will appear as orange in green background), see : Hooykaas, P. J. J., C. Roobol, and R. A. Schilperoort.1979. Regulation of the transfer of Ti plasmids in Agrobacterium tumefaciens. J. Gen. Microbiol. 110:99-109.).

2. Csaba propose you to use simply gentamicin resistance  (pMP90 has GenR marker). -nel, rathervthan any nopaline/octopine catabolism marker.

3.As a part ofthe he parallel deletin of the T-DNA region and its replacvement with  gentamicin rezisztencia gene the ornithin cyclodeaminaz gene (in NOC region) was knocked out. Consequently, thea pMP90 containing GV3101 strtain can not degrade neithervthe oktopine nor the nopaline.

I do not know (but intend to learn) whether the EHA105 or LH4404 strains could or could not cataboliye the respetives opins.

Michale, please give me e feed-back.

Regards, Andras Fodor

INFORMATION FROM CSABA KONCZ TODAY:

1. For selecting for opine catabolism in a minimal media zou should use BTB (bromethzmople-blue)  pH indocator (the opine/cataboliser colobnies will appear as orange in green background), see : Hooykaas, P. J. J., C. Roobol, and R. A. Schilperoort.1979. Regulation of the transfer of Ti plasmids in Agrobacterium tumefaciens. J. Gen. Microbiol. 110:99-109.).

2. Csaba propose you to use simply gentamicin resistance  (pMP90 has GenR marker). -nel, rathervthan any nopaline/octopine catabolism marker.

3.As a part ofthe he parallel deletin of the T-DNA region and its replacvement with  gentamicin rezisztencia gene the ornithin cyclodeaminaz gene (in NOC region) was knocked out. Consequently, thea pMP90 containing GV3101 strtain can not degrade neithervthe oktopine nor the nopaline.

I do not know (but intend to learn) whether the EHA105 or LH4404 strains could or could not cataboliye the respetives opins.

Please give me e feed-back.

Regards, Andras Fodor

This question is pretty old, but from the OP question and responses it sounds to me like they actually have E. coli in their stock tube, and probably not Agrobacterium tumefaciens strain GV3101pMP90. An examination of the color of cells pelleted from an overnight culture will very likely reveal the likely identity of the mystery organism. Remember, for most Agrobacterium strains with which we routinely work, it must be pink and it must stink!

I dont think that OP were so dull (mixing E. coli to Agrobacterium).

@Andras: I certainly didn't mean to suggest that the OP is dull. I think contamination is indiscriminate in labs, and is an easy thing to check and fix. That was my point. I had a similar thing happen to me a few years back, and it was due ultimately to an autoclave that didn't reach the proper temperature, resulting in a low level of contamination in the broth that escaped detection prior to use for growth of the Agrobacterium culture for preparation of electrocompetent cells.

Hi,

First of all, I appreciate all above suggestion, it was helpful at some point in my trying to transform agrobacterium for the first time. However, based on my experience with also getting bacterial lawn in my plates after less than 24h, I worked on diluting the concentration of the transformed plasmid. So, basically, I transformed approximately 0.1 ng of the destination vector which carrying my gene of interest and then plate less than 50 μl on media with 50 µg /ml rifampicin & same for Kanamycin. I'm using GV3101 strain of Agrobacterium and following the manufacture protocol. The result was good with nicely grown colonies so I proceed with that. One more thing to consider when optimizing the plasmid dilution is the source of your component cells. If you are using the one from a kit, it have high transforming efficiency than the one that be prepared in labs. Hopefully, my contribution is helping the readers.

For the agroinfiltration of Nicotiana plants, which si better GV3101 or GV3010?

Turn to Prof. Csaba Koncz [email protected]

+49 221 5052213