EGFP constructed in T-vector can express green fluorescence? - SOS (Dec/21/2007 )
Hi, everyone. I have encountered a very surprising thing. Recently I have constructed EGFP into T-vector (bought form TakaRa company). And the recombined T-EGFP vetcot was transfered into DH5a. Now on the LB solid medium I can see many green bacterium colony, which is different from blue spot. Does anyone know the reason? Thank you for any suggestion.
Why do you think you should ever see blue spots with an inserted EGFP gene? EGFP is green (the G in there is telling you that). Are you possibly confused about X-Gal plates and blue/white selection for inserts?
Thanks for your reply! But I am just confused about why the baterium colony was green and the white spots was so little. The EGFP gene in T vetor could be expressed in DH5a and could be seen without UV?
Yes, GFP expressing bacterial colonies look green, although they are much more visible with long wave UV.
But is the T-vector only a clone vector? Why could it express the eGFP? Will you please explain the principle?
I have no idea what you mean by "clone vector." You are inserting a GFP gene into a vector, transforming it, and getting green colonies. What part of this is surprising? If you are asking why it is being expressed, then this will depend on the promoters present in your vector or insert, but even without explicit promoters, it is not uncommon to see expression from cryptic promoters present in the construct.
Thank you very much for your kindly help. What I use is pMD18-T vector(TaKaRa company). This vector only exist a promoter for expression of Lacz gene. I could not find another promoter for expressing other inserted gene.
I have ever selected a green spot of bacterium to cultivated in liquid LB. But the result of restriction digestion showed that the EGFP gene wasn't inserted into the vector. I am so confused with the green spot! How were they produced?