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 Results

First we looked at controls and non-dronpa constructs to establish a baseline to compare with dronpa. The CheY knockout strain from the Keio collection displayed no swimming regardless of light stimulation, as expected. The CheY overexpression strain displayed tumbling regardless of light.

Now we could compare the behavior of chey dronpa with our wild type strain. For chey dronpa, we recorded 7 swimmers in the first trial. This is in accordance with the default dimerized state of Dronpa. After 500-nm light, CheY should be reactivated, which agrees with the decrease in swimming we observed in the second trial. In the final trial, we didn't see a resurgence in swimming as we would expect, and we are exploring the possibility that 400-nm light is toxic to E. coli.

Here we have a table summarizing all of our microscopy results. As you can see, we actually observed many of the behaviors we expected, including critically our Dronpa constructs This indicates that we were successful in controlling E. coli motility using Dronpa.

Swarm Plate Results

Alongside our phase-contrast inverted light microscopy assay, we also conducted swarm assays using semi-solid agar plates to observe motility behavior differences between our constructs.

In order to do this, we needed to create a light-emitting apparatus that was capable of shining the 400 and 500-nm wavelengths of light that would activate and deactivate our Dronpa proteins.

Our swarm plate data under 500-nm light resulted in approximately equivalent motility between our constructs with dronpa being monomerized due to 500-nm light exposure. In other words, the probability of running and tumbling was approximately equivalent between constructs.

95% Confidence Interval:

• WT CheY: .316-.684
• WT CheY Dronpa: .377-.648
• Mut CheY Dronpa: .445-.605
• DH5-alpha: .152-.648

However, this is not what we would expect in terms of motility because on the left, we observe bacterial growth that did not diffuse through the agar surface. On the right we have a picture that represents our first pilot of swarm plates that illustrates true bacterial diffusion through the agar, not just growth on the agar surface.

In terms of subsequent troubleshooting, we realized we had increased the agar concentration in the plates on the left, and it is possible that the incubation temperature changed as the plates on the left were exposed to more heat from the light apparatus. Repeating these experiments using lower concentration agar plates is an area for further experimentation to further characterize our system alongside our microscopy results.