I am currently running growth curves on several strains of E.coli containing plasmids harbouring venus. I am taking both OD600 and venus fluorescence measurements across an 18 hour period in a tecan plate reader. I am using a WT BD792 E.coli strain as my control for the fluorescence as it does not fluoresce with venus, however, on analysing the plate data the venus signal coming from the control is higher than any of the strains harbouring the venus bearing plasmid. The BD792 is being grown without antibiotic and the other strains are being grown in kanamycin 50 (everything is being done in M9 media). The OD of the BD792 strain does increase a lot quicker than the other strains but the venus signal is so high I can't believe it is just autofluorescence, the WT by itself hits the fluorescence detection limit of the machine. Any advice would be appreciated!
Thanks,
Jack
The "fluorescence" of bacterial cells expressing a fluorescent protein measured in a plate reader is a mixture of fluorescence from the fluorescent protein, autofluorescence of the cells, and light scattering by the cells. The amount of light scattering can be substantial and depends strongly on the cell density, as well as the shape of the cells. Autofluorescence depends on the growth conditions.
If you can, take excitation and emission spectra of the cells to check for a peak at the expected excitation and emission wavelengths of venus. If you don't see them, then the fluorescence is mostly coming from autofluorescence and light scattering.
To measure fluorescence in a plate reader use black plates, either with clear bottoms for bottom reading or black bottoms for top reading. Don't use clear or white plates.
Hi Adam,
I am using black plates with clear bottoms so I can also measure OD600 at the same time. I will check the spectra of the cells in another machine as the plate reader itself doesn't have that ability, thanks for the suggestion!
Jack
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence