We have tried transforming our Bacillus strain with a Tc containing GFP plasmid. The minimal inhibitory Tc concentration of the strain is 1 ug/ ml. After electroporation the transformants grow on 2ug/ml plates. It gives the initial picture of a successful transformation. Yet there is no fluorescence of any colony of the plate. The initial thought was Tc which is light sensitive. Yet the untreated wild type do not grow on the 2 ugml Tc plate indicating that even in case Tc light degradation still enough present for inhibition of wild type. Should we conclude that the plasmid is not expressed in the stain……Sure we could use PCR e.g. on Tc gene and see if the transformants contain the plasmid.
Yet If no fluorescence the tagging with GFP plasmid has failed anyway.
We need to tag the bacterium somehow since it is a good PGPR strain. Do you think some antibiotic resistance marker could be applied ? I would be grateful for any recommendations.
There was a cover photo on the Journal SCIENCE years ago that had variable expression of GFP in E coli grown in broth culture with all conditions being equal. The full spectrum of response from no fluorescence to very bright cells was represented. So even under ideal conditions, not all of your cells would likely express GFP. There may be a better marker, multiple antibiotic resistance might be better, just don't create a 'superbug' :-)
Thanks. I attach the paper with the pBSVG101 details used by us. The plasmid gives nice flourescence in E. coli, B. subtilis, P. polymyxa. Yes, we have checked the culture under microscope with equally missing results of amy fluorescence
This is interesting and curious:
1) Because you have shown that the plasmid fluoresces in other bacteria, you have ruled out a case of non-recombinant transformants. If you created the plasmid from scratch, you may have transformants (containing the vector) without the insert that will grow on the selective medium
2) Could this strain be affecting the post translational conformation? Or did the gene not get expressed?
3) If your goal was just to tag and track, double antibiotics marker will do; however if you are interested in following gene expression, antibiotic markers become cumbersome and difficult to interprete.
It is hard to transforming plasmid into bacillus, we spent over one year to achieve it. The problem is on the condition of electroporation, you need to optimize it. And there may also have some problem in the plasmid. Becasue most of wildtype strain contain numerous plasmid, and these plasmid may inhibit the duplication of the GFP-contain plasmid. So you also need to try more plasmid which were constructed by different species of bacillus.
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