Part:BBa_K325108:Experience
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Applications of BBa_K325108
Team:EPF_Lausanne 2014
Author: Sakura Nussbaum
Comparison of Firefly and Renilla full and split luciferases
Aim
Luciferase complementation assay is a powerful tool to study interaction between proteins in vivo. Thus, it is important to choose carefully which signal is best suited for these kind of experiments.
As an improvement of this biobrick, we submitted to new parts ( https://parts.igem.org/Part:BBa_K1486016 and https://parts.igem.org/Part:BBa_K1486017 ) that are the two moieties of the split EPIC Firefly luciferase.
Method
In the complementation assay experiments, it is important also to have good controls. As a positive control, the full luciferase was used, and as a negative control, the split without fusion protein was used.
In our experiment, we aimed to test which of the luciferases would best suit a complementation assay (in our case, we used the interacting CheY and CheZ proteins, see this page (https://parts.igem.org/Part:BBa_K1486054)). We compared our construct (https://parts.igem.org/Part:BBa_K1486022) to the part EPIC Firefly.
The protocol for our bioluminescence assay can be downloaded here [[1]]. The detailed results of this experiment are on this page http://2014.igem.org/Team:EPF_Lausanne/Results.
Results
As shown in the graph below, we could determine that Firefly luciferase was more suitable. For the same concentration of substrate, it has a more stable and higher signal. Moreover, the difference between the background noise (negative control, non fused split luciferase) and the full luciferase is bigger for Firefly luciferase, which is also preferable.
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