I have an M. smegmatis strain with GFP in pFPV27 vector(with kanamycin resistance) under the control of Mtb rrn promoter.When I transform this bacterium with pVV 16 vector(with hygromycin and kanamycin resistance) having my gene of interest, GFP expression is completely lost as observed by fluroscence/confocal microscopy and western blotting in comparsion to empty vector and some other gene cloned in pVV, transformed M.smeg.
Note-: In all of the different (transformed/untransformed) M.smeg expressing GFP, non GFP bacterium are also present, as observed by DIC(Differential interference contrast) and fluorescence microscopy.
What could be the possible reason for loss of GFP expression?
Thank you.
If I'm not wrong, the original pFVP27 has a kanamycin resistance, while the pVV16 should have a hygromycin resistance (derived from pMV261, I thought replacing the Kana R), so in theory using the double antibiotic selection you should be able to keep them both, although sometimes, as far as I know, the Oris are not compatible.
But if the situation is as you say, with the pVV16 containing both selection markers (I might well be wrong about the plasmids, I'm not really into this field!), it is difficult to keep both plasmids in the cell, because one is sufficient to provide survival, so I would expect the GFP vector to be progressively lost...
@Pietro Pilo Boyl -:thank you for your answer.
even I fell so, but there is complete loss in M smeg with my gene of interest and not with M.smeg transformed with pVV containing someother gene, that is what, is amazing me
Results of your experiments suggest that the plasmids are compatible and GFP is non-toxic for M. smegmatis. In the presence of the plasmid encoding your protein of interest the GFP protein is lost. I think the matter is in your gene or protein of interest. There are several stages of GFP expression that your protein may affect.
1. Your protein is a transcription factor that directly or indirectly represses transcription from promoter that is used for GFP gene expression.
2. If this protein can interact with GFP and, on the other hand, interact with some ATP-dependent proteinases (like Lon-proteinase) or other proteases, thereby recruiting it for proteolysis. This interaction can take place co-translationally, when GFP is susceptible for proteolysis.
3. Your protein affects folding of GFP directly or indirectly. Again active protein can not be formed and in since it is misfolded it is a good substrate for proteinases.
4. Your construct contain hidden ORF that interferes with GFP biosynthesis or proper folding.
If your protein is not supposed to do any of that is mentioned above, but it is a heterologous protein form other organism it is possible that it can change its function or have some function that is not described in literature yet.
You can easily discriminate between transcriptional and post transcriptional (cotranslational) effect or your protein by measuring mRNA level of GFP by RT-PCR.
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