Team:BS United China/Proof Of Concept

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Proof Of Concept

The previous section is a detailed explanation of where we see our project being implemented into the real world. Despite various discussions and interviews were conducted to ensure the most reliable conclusions, those conclusions were after all, only theoretical ideas. We can’t merely be armchair strategists and experimenting is the only method we can test whether our fundamental concept can be proved.

That being said, our team agreed in full accord that PVDQ is by far one of the most crucial component of our entire project. It’s directly responsible for suppressing the growth of P. aeruginosa. Therefore, in response to the proof of concept, we designed 2 additional experiments to verify the suppressing function PVDQ has.

Experiment 1: Simulating the effectiveness of PVDQ in
suppressing P. aeruginosa in food

This experiment was conducted on spread plates. We used LB culture to simulate food and spread OD₆₀₀ P. aeruginosa solution (5000x diluted) evenly on the surface of it. Then, 50μL of PVDQ is also spread on the surface to test its suppressing effects.

Three spread plates with only P. aeruginosa spread but no PVDQ were also created and observed as the control group, and three spread plates with PVDQ applied were used as the experimental group.

To create 100mL of LB culture:

- 1 gram of sodium chloride.

- 1 gram of tryptone.

- 0.5 gram of yeast extract.

- 115μL of 4M sodium hydroxide.

- 1.5 gram of agar.

Fig.1 Control group after cultivation

Fig.2 Experimental group after cultivation

Above is the 6 control and experimental group spread plates after P. aeruginosa was allowed to cultivate.

Fig.3 Calculating the number of bacteria strain in each spread plate

We counted the number of P. aeruginosa strain grown by dividing each spread plate into four even sections then counting the number of strains in one of the sections, the number is indicated at the top left on each plate. Then, we multiply that number by 4 to calculate the average total number of strain grown in the control and experimental group.

Control Group Average: 992
Experimental Group Average: 372

As shown by the results, the number of strain growth in the three spread plates with PVDQ added is significantly less than that of the control group, indicating the PVDQ is effective in suppressing the growth of P. aeruginosa.

Experiment 2: using a spectrophotometer to test the effectiveness of PVDQ in suppressing the growth of P. aeruginosa and drawing growth curves

This experiment was actually among the very first we conducted in the lab. The main idea was to have different amount of PVDQ added to P. aeruginosa and using a spectrophotometer to measure the OD value to determine concentration of bacteria.

Fig.4 Testing bacteria concentration using spectrophotometer

Four different groups were used: control group with no PVDQ added, and E1, E5, E8 as experimental groups. They respective represent the number of times PVDQ is eluted before added to P. aeruginosa.

The experiment lasted for 12 hours and we tested the four groups on an hourly basis. While not being tested, the four beakers were placed in shaking incubators to ensure the steady and un- interfered growth of bacteria.

Fig.5 Results table of the 4 different groups

The unit we used was OD600nm, and from the results table it’s fair to say that for the initial 6 hours, the control group with no PVDQ added showed significantly greater growth of bacteria than any of the experimental group. However, after the 7th hour, all three experimental groups showed drastic increase in their OD value. We concluded that this is due to a lack of amount of PVDQ used, and the fact that PVDQ becomes ineffective after a lengthy period. Nevertheless, the fact that the OD value almost quadrupled immediately after PVDQ became ineffective also indirectly shows the suppressing function it has while effective.

Fig.6 The comparison of the growth curves of the P. aeruginosa after treated with PVDQ proteins

From the eventual growth curve we can also see that up until the 7th hour all three curves that represented groups treated with PVDQ proteins remained under the control group.

In conclusion, both of the experiments demonstrated successfully the fact that PVDQ protein has a solid effect in suppressing the growth of P. aeruginosa. The results were promising as it not only showed convincing evidence that our project’s concept can be verified in reality, but also pointed out potential flaws in our design that could be modified. For example, working on elongating the effective time for PVDQ proteins.