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detection of GFP fluorescence in whole cell bacteria - (Feb/24/2006 )

Can anybody help me in detecting the GFP Fluorescence in whole cell bacteria? I have cloned a Green Fluorescent protein (GFP) gene into vector under the heavy metal inducible promoter, the principle is whenever that particular heavy metal is supplied in the media, it should induce the transcription of GFP gene producing green fluorescence under UV light. After induction we used to visualise the green fluorescence in Axiocam Fluorescence microscope (Zeiss), we have also tried to quantify the green fluorescence by Mithra's Fluorimeter, the problem is earlier we got good differentiation between negative control and induced cells, but when repeated the same experiment the values were same for both, another problem is that while using Fluorecscence microscope the negative control also looks green cool.gif , thatswhy I was little bit confused, whether the GFP is actually fluorescing? How a E.coli bacteria will look under Fluorescent microscope fitted with GFP filter lense (perfect white or green tinge), if anybody knows how to analyze the GFP Fluorescence in live whole cell bacteria, please let me know

thanking you in advance smile.gif



It sounds like you are getting expression from your "negative" control. Maybe there is enough heavy metal contamination in your medium. You desparately need a more negative control: untransformed E. coli of the same strain you transformed. This will be guaranteed not to have GFP expression. The good news is that you already have them.