I just want to know if some of you already had this problem and if you have some tips to solve it :
I tagged succesfully my mucoid bacteria because i see under microscope they expressed the GFP but, when i look my plate with colonies under UV light, it is not so evident to see the GFP whereas for other strain it works well.
This strain is really viscous, probably due to an overproduction of alginate or other EPS, that may block the GFP fluorescens (I find nothing related to that...).
I do not want to use plasmid tag which may amplify the GFP signal, but really a chromosomic one.
Thanks.
I just verify the fluorescens under an epifluorescent microscope, because i saw nothing by just putting my petridish plate with colonies under uv light
Dear Jordan,
A few questions for you:
What is the emission spectrum of your UV lamp? Is it the one you use to visualize agarose gel bands with ethidium bromide stain?
What is the excitation spectrum of GFP?
Is there significant overlap?
Please post your findings to help other people...
Yes, it is the UV lamp use to visualize agarose gel. This technique worked well with other closest related strains using the same GFP-tag (MiniTn7-gfp2).
Basicaly under the microscope : Excitation 470nm, Emission filter of 505–530nm. I think that around 60% of the cells expressed the gfp.
Will the different GFP variants (such as GFP vs. EGFP) work the same good under UV lights? I think the optimum spectrum for them are different??
I don't know but i think the EGFP is just a question of protein stability, and the emission/excitation wavelength may not differ...
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