Team:Tsinghua/Proof of Concept
Proof of Concept
The effectiveness of our engineered bacteria was verified by the degradation analysis of biofilm. We used Nikon A1R HD25 confocal laser scanning microscope and NIS Elements AR software to visualize the process of biofilm being degraded by the engineered bacteria in 3D and planar images and movies.
The biofilm degradation experiment using engineered bacteria:BL21-NOS
We use the constructed BL21-NOS and with inducer IPTG to degrade the biofilm. PAO1 biofilm with GFP fluorescence signal is continuously observed under the confocal microscope for 3h. We randomly select three fields in each divide of the petri dish.
In the petri dishes with our engineered bacteria, we observed biofilm significantly decreased. The 3D movie shows the degradation behavior of one large lumpy biofilm degradation. We can see the biofilm became smaller and dispersed gradually (the dispersed bacteria can be seen).
The 3D movie of PAO1-GFP biofilm degradation under confocal (with BL21-NOS)
As the planar movie (in one of the selected field, others have the similar effect) shows, we can clearly see that the green lumpy biofilm fluorescence signal in the middle of the field of vision gradually decreases with time, and the surrounding smaller biofilm gradually disappears and becomes a cloudy dispersed bacterium.
The planar movie of PAO1-GFP biofilm degradation under confocal (with BL21-NOS)
And we select three time point: 0h,1.5h,3h for image analysis. We further confirm the effect of degradation using GFP signal area analysis and calculate the GFP area each time point/ original GFP area(0h). As the line chart shows, the GFP area of the biofilm is gradually decreasing.
The three-time-point image and GFP area line chart shows PAO1-GFP biofilm degradation (with BL21-NOS)
We also set two groups of control to check the biofilms original behavior and exclude other possibilities (two control groups: M9 w/o IPTG and substrate ). PAO1-GFP biofilm with GFP fluorescence signal is continuously observed under the confocal microscope for 3h. We randomly select three fields in each divide of the petri dish. The behavior of the biofilms in control groups are all similar: the middle biofilm signal remains (or decreased a little bit). As time goes on, many small biofilm grow back around and do not disperse. This indicates that the biofilm is still forming and growing normally in the control group.
The control group’s biofilm growth behaviour
We further confirm this using GFP signal area analysis and calculate the GFP area each time point/ original GFP area(0h). As the line chart shows, the GFP area portion of the biofilm stays about the same level and even went up in the third hour.
The three-time-point image and GFP area line chart shows PAO1-GFP biofilm area level remains and even many small biofilm emerged (control)
With the time-lapse 3D and planar confocal imaging, we could say that our NOS-expressing engineered bacteria works in degrading P.aeruginosa biofilm.
Reference:
[1]Reichhardt C, Parsek M R. Confocal laser scanning microscopy for analysis of Pseudomonas aeruginosa biofilm architecture and matrix localization[J]. Frontiers in microbiology, 2019, 10: 677.
[2]Roy A B, Petrova O E, Sauer K. The phosphodiesterase DipA (PA5017) is essential for Pseudomonas aeruginosa biofilm dispersion[J]. Journal of bacteriology, 2012, 194(11): 2904-2915.
