iRFP proof of concept
iRFP was utilised as a reporter in our project. Having correctly cloned in iRFP to our plasmid of of interest pNZ44, we had to determine if it was functional. Initially we had thought that the required co-factor, biliverdin, was only available in the cytosol of the RAW 264 cells. However, we subsequently found out in our experiments that it was in Foetal Bovine Serum (FBS) as well.
Therefore it was now possible to validate whether our protein is functional in the presence of the cofactor biliverdin.
To do this, we compared L.lactis expressing iRFP in media with FBS and media without FBS across several concentrations of L.lactis (written in terms of the corresponding multiplicity of infection used in the “Real World Demonstration” experiment. Hereafter referred to as MOI for simplicity). L.lactis was grown in media with FBS for 4 hours to allow for the protein being expressed to interact with biliverdin which it uses as its phytochrome. Following this, the plates were analysed using the Odyssey infrared scanner. (Fig. 1 and 2). The fluorescent intensity was also plotted against the multiplicity of infection. (Fig. 3).
Fig. 1: Photographs of L.lactis in FBS at three different MOIs in media with FBS visualised using Odyssey infrared scanner. Left to right: MOI=100, MOI=300, MOI=1000.
Fig. 2: Photographs of negative control plates (L. lactis in media without FBS).
Fig. 3: Graph of fluorescent intensity against MOI. There is a linear relationship between the amount of L.lactis initially aliquoted to each well and the fluorescence intensity at the end of the experiment. This is reflects the fact that, as the number of L.lactis cells increases, a greater overall amount of protein is produced. The plates containing the L.lactis with FBS in the medium showed fluorescence under the infrared scanner, and the resulting graph of fluorescent intensity against MOI shows that there was a linear relationship between the two. There was no fluorescence observed from any of the wells of the plates containing no FBS.
The plates containing L.lactis with FBS (and hence biliverdin) showed fluorescence, while those with no FBS (and hence no biliverdin) showed no fluorescence. This is proof that the iRFP is functional in the presence of its co-factor, biliverdin.