Team:FJNU-China/Demonstrate
Demonstrate
Overview
In this project, our team focused on inhibiting the harmful bacteria and bad smells in our daily life. Using the method of synthetic biology, we successfully produced phenyllaclic acid (PLA) with broad spectrum bacteriostatic function and rose-aromatic compound 2-phenyl alcohol (2-PE) by the recombinant E. coli. We think it has the potential application in laboratory and household usage. Our project inspired us to propose some specific applications of our rose-scented bacteriostat, including in waste beaker in the lab and axilla. We believe that our work has reached the medal requirements of demonstration as we have confirmed the function of PLA and 2-PE and made sure our engineered strains can produce those two products. In addition, we also have demonstrated our waste beaker cover in the laboratory to prove the practical feasibility of our products. The details of the demonstration are shown below. You can learn more details of the experiment and results in our design and results, modeling page.
Part1
Validation of Broad spectrum antibacterial function of PLA
Part2
Practicability and Mechanism of PLA
Part3
Prove the practical feasibility of our products
Part4
Demonstrate the advantages of our products
Part1: Validation of Broad spectrum antibacterial function of PLA
We have tested the effects of PLA on a variety of bacteria, and measured the residual amounts of bacteria over time to verify the broad spectrum antibacterial properties of PLA. Because of the limited strains in our laboratory, we completed this part in cooperation with other teams. We measured the inhibition effect of PLA on yeast Cen.PK2-1C, Staphylococcus aureus, Pseudomonas aeruginosa, yeast Cen.PK2-1D and other bacteria respectively (For details, please read the Collaboration page). The collective data figure is as follows.
Fig.1 Growth of different initial value of OD Cen.PK2-1D under 16 mMol/L PLA
Fig.2 Growth of different initial value of OD Cen.PK2-1C under 16 mMol/L PLA
Fig.3 Growth of different initial value of OD Staphylococcus aureus under 0 mMol/L PLA, 10 mMol/L PLA, 15 mMol/L PLA, 200 mMol/L PLA.
Fig.4 Growth of different initial value of OD Pseudomonas aeruginosa under 0 mMol/L PLA, 10 mMol/L PLA, 15 mMol/L PLA, 200 mMol/L PLA.
From the above data, we can clearly see that PLA can inhibit the growth of most bacteria, which directly proves its broad-spectrum antibacterial properties.
Part2: Practicability and Mechanism of PLA
Phenylalanine is converted to phenylpyruvate by the action of an aminotransferase (Tyrb) from Escherichia coli 21B, which is then dehydrogenated by lactate dehydrogenase (D-ldh) to form phenyllactic acid (PLA). Through the above experimental methods in part1, we simply qualitatively analyzed the antimicrobial effect of PLA, and subsequently we further verified the antimicrobial effects and practicability of PLA through modeling and experiments. (For details, please see the Model page).
（1）Quantitative Validation of bacteriostatic effect of PLA
We illustrated the effects of different concentrations of PLA on Staphylococcus epidermidis with different initial value of OD, and we measured the growth curve every hour and obtained the formulas and diagrams below.
Fig.5 Simulation of the number of bacteria colonies under different concentrations of PLA.
Staphylococcus epidermidis is the main cause of armpit odor. From the above chart we can see that PLA has an effective inhibiting effect on Staphylococcus epidermidis, and the higher the concentration of PLA, the better the inhibition effect, which quantitatively proves the effectiveness of PLA and the practical feasibility of the products we used to deal with armpit odor.
（2）Timeliness of PLA inhibition effect
Fig.6 Growth of saphylococcus epidermidis with an initial OD value of 1 under 11.25mmol/L of PLA
In addition, we determined the optimal PLA concentration to inhibit Staphylococcus epidermidis, at which the growth rate of bacteria was always lower than the mortality rate. When PLA is higher than this concentration, the reaction rate will be faster.
（3）Validation of the breadth applicability of specific PLA concentration（Mechanism of PLA）
Fig.7 Growth of staphylococcus epidermidis with an initial OD value of 0.5 Abs under different concentrations of PLA
Fig.8 Growth of staphylococcus epidermidis with an initial OD value of 2 Abs under different concentrations of PLA
Through these two figures, we learned that the effect of PLA in inhibiting the bacteria is independent to the initial OD value, which means the certain amount of PLA have the same inhibiting effect on the different amount of bacteria. The above analysis showed that under the same concentration of PLA, the number of strains inhibited by PLA is in proportion to the total number of current strains.
Part3: Prove the practical feasibility of our products.
This year, the main focus of our project was to prevent harmful bacteria in the laboratory waste beaker and to solve its odor problem. In addition, we designed a waste beaker cover to achieve the desired effects. In order to verify the practical feasibility of our products, we applied this waste beaker cover to our laboratory to test its function. We set up some contrast experiments using two beakers in the lab. The beaker contains the experimental waste produced by our usual experiments, including the pipette tip, the paper scraps, the discarded tubes, and so on. We applied our cover to one of the beakers and pressed the red button to release our engineered bacteria which produced rose-scented bacteriostat, and the other beaker was left untreated as a control. We exposed them to the same environment. After 24 hours, they had subtle differences. And we observed a more obvious comparison (as the figure shown) after 3 days. In the natural environment, the untreated beaker was filled with white colonies; on the contrary, there is less bacteria in the beaker with PLA. Regarding the odor contrast, the beaker of the blank control gave off bad smells, while the other beaker showed a noticeable improvement with a faint rose scent.
Fig.9 The result of demonstration experiment
In summary, we have demonstrated our design in the laboratory, which proves that our antibacterial and aroma substances can solve the problem more effectively of harmful bacteria and odors in the experiment.
Part4: Demonstrate the advantages of our products.
On the other hand, we conducted a survey of similar products on the market to demonstrate the advantages of our products, and tried to compare some antibacterial products in the domestic market with our rose-scented bacteriostat. We chose a hand sanitizer product with a fresh fragrance and a representative soap as a control. We firstly prepared 10 mmol/l of PLA and dissolved 5 grams of the two bought products into 45g of sterile water. After we exposed the prepared plate in the laboratory for 12 hours, we added 100 ul of three products prepared in advance on the surface. They were observed after 12 hours of incubation in the same environment.
Fig.10 The result of control experiments with similar products on the market
The No. 1 plate showed the growth of the bacteria under normal conditions. The No. 2 plate was treated with soap, and the third was treated with hand sanitizer, and the No. 4 plate was coated with our rose-scented bacteriostat. As shown in the figure, we can clearly see that the number of colonies on the No.2, No.3, and No.4 plates is reduced compared with the No. 1 plate. Among them, the soap has a relative weaker antibacterial effect than the other two products. And the hand sanitizer is equivalent to our antibacterial and aroma-producing substances. Our rose-scented bacteriostatic and the hand sanitizer have comparable bacteriostatic effects. In addition, we produce such rose-scented bacteriostatic, reducing the burden on the environment and being more friendly to people use the methods of synthetic biology. In conclusion, we have demonstrated the advantages of our products can meet the people's requirements in our daily life.
Reference
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