Team:Penn/results/luminometer/

From 2011.igem.org

Luminometer Experiments | Penn iGEM 2011

The luminometer is a photon counting device capable of performing ultrasensitive bioluminescent and chemiluminescent assays. The output, given in relative light units (RLU), is a number that can be compared against a background value to determine the relative change in the number of photons being measured up by the device. We used the luminometer to test and characterize our luminescent proteins (Aequorin and Renilla), as well as test if our pre-ceolenterazine construct was working.

In all luminometer experiments, our HEK293T cells were transiently transfected with our gene of choice. During the day of the assay, they were lysed with a passive lysis buffer from Promega and scraped open. The lysed cell solution was then added to the luminometer. The RLU was measured both before and after the addition of any needed substrates or cofactors, and these results were then analyzed.

Our first luminometer experiment was to verify that our Aequorin protein was properly expressing. We added the lysate from Aequorin cells to the luminometer and measured the RLU of Aequorin, Aequorin with Calcium added, and Aequorin with Calcium and Ceolenterazine added (Figure 1). As expected, the addition of only Calcium did little to increase luminescence, but the addition of ceolenterazine increased the RLU by about 5 orders of magnitude.

After verifying that our Aequorin is properly expressing, we wanted to test the kinetics of the Aequorin luminescence by varying the order in which Calcium and Ceolenterazine was added. In Aequorin that was preloaded with Ceolenterazine, the addition of Calcium into the cell lysate caused a rapid spike in luminescence followed by an equally rapid decay. Adding additional ceolenterazine caused an additional spike that was greater than the calcium spike. After 1 minute, the signal was still increasing, which allows to conclude that the Aequorin-Ceolenterazine binding is the slow step of the reaction (Figure 2).

We negatively controlled for Aequorin by using untransfected HEK239T cells. The addition of Calcium added no luminescence to the sample. The addition of Ceolenterazine added some luminescence, but the increase was so slight that we could conclude that our Aequorin was functioning properly (Figure 3).

Besides our Aequorin, we tested the expression and function of our Renilla luciferase protein. Following the same protocol as we used for Aequorin, we measured the RLU of our Renilla luciferase both before and after the addition of the Ceolenterazine. The signal after the addition of Ceolenterazine was 7 orders of magnitude greater than the basal level of Renilla luciferase (Figure 4).

Finally, we tested to see if we could get our Preceolenterazine to function properly with our Aequorin and Renilla. This would allow our sending and receiving cells to be completely self-sustaining – no input of ceolenterazine would be needed to start the signal. We took cell lysate from cells that were transiently transfected with our PCZ gene an added lysate from cells expressing Renilla luciferase. There was no increase in luminescence (Figure 5). We then took the PCZ lysate and added it to Aequorin lysate that was preloaded with Calcium, which again yielded no signal. When adding CZ to the same Aequorin sample, the RLU spiked dramatically, indicating there was a problem with our PCZ (Figure 6).