Well Upendra...seems your problem is solved according to this paper....it says ccdB is non toxic to Agro...

http://www.plantmethods.com/content/7/1/42

A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens

You may want to give it a try....

Hi Uprendra,

what about just a piece of DNA cloned into pDEST, which peferably does not interfere with gene expression of your host / destination organism....?

I wonder anyway for what purpose do you need an empty vector control? With gateway cloning there should be a lot of control constructs already.( you are using Agrobacterium mediated random insertion, right?). Make sure that you get several independent(!) lines from every construct.

Have fun

Best wishes

Lars

Hi Lars,

Thanks for your suggestions. I wanted to include empty control since i thought this is how the reviewers want to see the experimental design. May be i am wrong. Yes i could try to include some random gene as empty vector control (but that doesn't sound like empty vector. Isn't it?). Anyway i like your idea of using several independent insertion lines for every construct. 

Cheers,

Upendra

Hi Upendra,

[Response to your above last post:]

1. The purpose of including 'empty vector' in your experiments is to evaluate if the elements on the 'empty vector' alone (without your inserted gene cassette) will affect the transgenesis result, a non-specific effect.

2. If you add a random gene into your 'empty vector', it is not 'empty' anymore. Because, by using this vector, you cannot tell the final transformation results is due to the gene cassette you inserted in or due to the random gene or backbone alone.

3. Besides, to randomly add a gene into your empty vector and include it in the dataset may cause confusion to the paper reviewers in the future.

4. The ideal empty vector should be the vector without your gene-of-interest cassettes. Although many transgenic papers did not include an empty vector in their experiments, it always make your results more precise and your experimental design 'more professional' if you include an empty vector as a control.

Hi Yuan,

Thanks for clear and concise explanation to my problem. I will try to figure out a way to use the destination vector containing ccdB as empty vector.

The way the gateway system works is that a gene of interest is placed inside the cassette of ccdB rendering it thus inactive. Using ccdB as is is indeed a problem, Upendra you are right.

I'm not sure about the details in that case but I would suggest you place your gene of interest but with one or more added STOP codons at the beginning of the cassette, so the whole construct looks pretty much the same as RNA is transcribed but is simply not translated.

Well Upendra...seems your problem is solved according to this paper....it says ccdB is non toxic to Agro...

http://www.plantmethods.com/content/7/1/42

A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens

You may want to give it a try....

Thanks Sibaji. I will take a  look at the paper.

Hi Sibaji, I come into the same dilemma but thanks to your reply i know that ccdb is not toxic to agrobacterium. So I'll multiply my plasmids containing ccdb gene using a ccdb-tolerant stain like DB3.1 and then transform the plasmid into agrobacterium. Problem solved. Thanks. :)

Upedra and Jiexi what is your experience then ccdB is toxic to agroacterium or not?

Stupid question- but is there experience out there that the ccdB cassette wouldn't be toxic in the transformed plant either?

We are able to successfully transform ccdB containing gateway vector into agrobacterium strain LBA1100

Hi , I am facing the similar problem. What would you suggest for a Ecoli strain which is resistant to ccdB gene?

Dear Tejas Ra, You can use ccdB containing empty vector as control, Agrobacterium is resistant to ccdB and there is no problem in transforming ccdB containing plasmids to Agro an plants. Hope this solve your problem.