Using PEST to reduce background fluorescence

In our original experiments, there was a lot of fluorescence when our target molecule, theophylline, was not present. We hypothesized that this is because of accumulation of the highly-stable fluorescent protein. In an ideal system, this background fluorescence would be virtually nonexistant, as this would make interpreting the result of our test much easier. In order to reduce background fluorescence, we initially tried to use a Gal1 inducible promoter, so that fluorescence would not be "turned on" until the assay is ready to start. However, this did not reduce background fluorescence.

So instead, we tried adding a PEST sequence to the end of our YFP gene. PEST is a peptide sequence that can be tacked on to a protein in order to target it for degradation. With PEST, our YFP was targeted by the ubiquitin ligase pathway, leading to degradation by the proteasome.

This succeeded in reducing background fluorescence! As seen below, it is much easier to visibly see the difference between the theophylline and no-theophylline states with the PEST sequence than with the original construct, even though the fluorescence in the PEST construct is dimmer overall.

 

It can also be seen from this that as concentration of theophylline increases, so does fluorescence.

We quantified fluorescence using a flow cytometer. As seen below, overall fluroescence is much lower for the construct that includes PEST. Interestingly, this indicates a disparity between the way a flow cytometer measures fluorescence and how fluorescence is perceived by the human eye. While the difference between the theophylline and no-theophylline states is larger for the original construct, it is easier to visibly see this difference with the modified PEST construct.

 

Now that we have found a way to reduce background fluorescence, we can work on optimizing our system to make it more accurate and more sensitive. We can also use what we have learned from this proof-of-concept system to develop an aptazyme-based system to detect for shellfish toxins. Stay tuned for more on how this can be done!