I transformed agrobacteirum (LBA4404) cells with pGREEN-GFP vector but the GFP doesn't seems to be expressing in the LBA4404 cells, what is the problem? Why I am not getting any GFP expression? Is that a promoter problem or something else? Alo let me know about the promoters under which GFP expresses best in LBA4404.
Do you have a map of the vector you constructed? May I see it? Depending on the promoter you use, the expression may not be strong in bacteria... I assume you're using the 35s promoter. I use to work with lba4404 and gfp (pCAMBIA1304), and I can visualize gfp expression under UV, but the expression can be quite weak.
I do not currently have the vector map, its with my supervisor, i couldn't get any expression under UV light, this means I have to give some time to colonies to grow more in size or should I proceed to the restriction digestion confirmation step?
If you did not edit the plasmid, the GFP is under 35S in the pGREEN series (http://www.pgreen.ac.uk/a_cst_fr.htm). The idea is that the GFP is expressed in plants, not in the bacterium. The 35S is leaky in agro, that's why you may see some fluorescence. Note that the GFP excitation maximum is 395nm (long wave UV), and normally transilluminators for DNA are 310-365nm, so make sure to select 'long wave UV' if you can.
I'd suggest you test the bacteria by PCR or restriction digestion, and then do transient transformation in tobacco leaves to confirm that the GFP is working fine before moving into the real transformation. With transient you can see results in 3-5 days and move forward confidently. Otherwise, if you are not sure about GFP expression you are risking weeks-months of work.
Cheers,
Ruben
Thanks Mr. Ruben, i think his much information given by you is enough for me to understand its easily. Thanks a lot.