GFP expressing cells - masking of GFP expressing cells!!! (Aug/14/2006 )

Hi,

Can anyone please tell me if "non-gfp expressing cells have any masking effect on gfp expressing cells"......

Thanks...

I don't know exactly what you mean.

non transfected cells can grow faster than transfected cells, and then you dilute more and more your transfected cells, until losing them. Is that what you mean?

The objective is to create/develop a genetically modified natural E. coli isolate, using the lambda recombineering system that carries a gfp variant in the chromosome.

The non-gfp expressing cell (natural E.coli isolate) will be transformed with a gfp gene (as a PCR product). The non-gfp expressing cells carry lambda plasmids which aid in homologous recombination. The gfp gene will be integrated in the chromosome of the non-gfp expressing cells. The PCR product is “gfp gene flanked by 40bp overhangs from the homologous region from chromosome of the non-gfp expressing cells.

However, the concern is that after transformation of the non-gfp cells with the PCR product, the chances of the gfp gene being integrated into the chromosome might be less (i.e., less # of transformants). Therefore, to observe the transformants (gfp expressing cell) it is necessary to know if the non-gfp expressing cell (natural E.coli isolate) will have any masking effect on the transformant
Thanks.

Can you put a selection pressure with antibiotics to eliminate the non-transformants?

no, because I am going to release this GMO into the fields and the EPA does not allow to add any antibiotics.....

well in that case, only colony selection can tell you if you have the right clones, and you have to be sure that the gene stays in E coli over time. You are kind of playing agaisnt odds here.

Yes! i agree. in my case selection is not the issue (fluorescencing colony itself is the selection here)but i wonder there might be any masking effect on the transformants by the non-transformants. since here the transformant fluoresces (gfp gene in the chromosome)