Team:Pittsburgh/Results

Fluorescence Results

Dronpa is a photoswitchable fluorescent protein that loses fluorescence under 500-nm light and restores fluorescence under 400-nm light. In our fluorescence assays, we wanted to assess if our constructs are expressing Dronpa.

Plate Reader Fluorescence Assay

Since Dronpa fluoresces similarly to (in the range of) GFP, we looked at the fluorescence of each of our strains using the GFP settings of our plate reader. The Dronpa containing constructs fluoresced more than their non-Dronpa counterparts, which confirms expression.

Induction of Plasmid Promoter via IPTG for Dronpa Expression:

Under the phase contrast microscope, these images represent the induction of the promoter controlling the expression of the dronpa protein attached to a WT CheY molecule within our CheY KO cells from the Keio Collection. As the concentrations of IPTG increase, the fluorescence of the Dronpa protein, which is a GFP analog, increases proportionally. This demonstrates that the protein is being expressed and was successfully transformed onto CheY molecules within our engineered plasmids.

Microscopy Videos

In order: 1. Pre-illumination 2. After 2 minutes 500nm light 3. After 3 seconds 400nm light

CheY Knockout



CheY Wild-type



CheY Wild-type Dronpa Fusion



CheY Mutant