How to quantify GFP fluorescence from a bacteria? - (Oct/16/2007 )
Having trobule finding a good method for this quantification assay.
Question:
"You have constructed a plasmid vector containing a fluorescent marker gene (GFP) controlled by a constitutively expressed promoter, which you insert into a bacterium that you intend to monitor in a complex environment... You need to quantify the GFP fluorescence emitted by the bacteria grown in broth without selective pressure (a specific antibiotics) for a number of days in order to find out whether the fluorescence intensity decreases over time. What kind of device do you chose to use and why?"
I was thinking quantitative real-time PCR, is that correct or are there other methods that are better to use?
Question:
"You have constructed a plasmid vector containing a fluorescent marker gene (GFP) controlled by a constitutively expressed promoter, which you insert into a bacterium that you intend to monitor in a complex environment... You need to quantify the GFP fluorescence emitted by the bacteria grown in broth without selective pressure (a specific antibiotics) for a number of days in order to find out whether the fluorescence intensity decreases over time. What kind of device do you chose to use and why?"
I was thinking quantitative real-time PCR, is that correct or are there other methods that are better to use?
Hi,
Quantitative PCR will allow you to measure expression of the GFP gene with specific primers and Sybr green or a fluorescent probe. To do this you would have to purify the RNA then make cDNA and do the real time assay for each timepoint.
The question points towards the answer you want i.e. "You need to quantify the GFP fluorescence emitted by the bacteria" based on this try to come to an answer.
Best wishes,
Ceri
Thanks alot!
What about a spectrofluorimeter or flow cytometry (or is that the same thing?). Would that be an easy way to quantify the GFP fluorescence?
What about a spectrofluorimeter or flow cytometry (or is that the same thing?). Would that be an easy way to quantify the GFP fluorescence?
a spectrofluorimeter is a device for reading fluorescence. it is a component of other devices.
look up "flow cytometry" in wikipedia. this will give you a clue.