Team:NCTU Formosa/Safety

Safety

This year Apollo designed a product to mainly detect antigens. Our users include scientific researchers, clinical scientists and so on. We believe these people all have the expertise. On the other hand, our product contains E. coli so when applying our product, basic protective gear—lab coat and gloves—are required. Objects that come into contact with our product, for instance, tips, need to be sterilized and thrown away, in case of non-natural gene outflow. We will hand out our product to others after our safety procedure. Hence lowering the possibility of bio-contamination.


We designed a safety mechanism for our E.Cotector allowing it to be friendlier to users and to the environment. Our main objective is to maintain the fluorescence but kill the bacteria, which means that treated E.coli cannot grow in nutritious conditions such as LB.



Antibiotic

We tried antibiotics to achieve our goal. We used three kinds of antibiotics. The first one is tetracycline. It can bind to 30S subunit of ribosomes and then inhibit the synthesis of proteins. The second one is ampicillin. It can inhibit the formation of the cell wall. The third one is sulfonamide (p-Aminobenzenesulfonamide). It competitively inhibits the synthesis of folate, which connects to the purine synthesis and the DNA synthesis.



Method and Result

We mixed the antibiotics and bacteria together. We used E.cotector expressing both anti-VEGF and red fluorescence protein to do the test. After we added different concentrations of different antibiotics, we observed the fluorescence intensity and the bacterial growth on LB plate every hour. We extracted 100 µL of bacterial liquid from each sample and added it to 96 well to detect the fluorescence. There will be more details on this in the protocol.


In the experiments, after adding tetracycline, Ampicillin, we discovered that the fluorescence remains close to the original value (Figure 1.) . We also used sulfonamide. However, the solubility decreased causing the concentration of sulfonamide to distribute unequally. This may be due to the fact that sulfonamide’s solubility is highly related to the solution’s pH value. The lower the pH value, the better the solubility of sulfonamide. This also means that sulfonamide creates more damage to the structure of protein, causing the fluorescence to disappear.




Figure 1. Fluorescence of antibiotics treated E.Cotector are as large as untreated E.Cotector.

The second step, after the addition of tetracycline, we extracted bacterial liquid from each sample and added it on to the LB plate each hour, and cultivate them at 37℃. Once there were bacterial colonies, we determined that the antibiotics did not fully kill the bacteria. Experimental results showed that when we added Ampicillin to the bacteria, bacteria could still grow on LB plate as untreated ones. On the other hand, bacteria added with Tetracycline will not grow on the LB plate (Figure 2.) .


According to (Figure 3.), we can found that the growth of our E.Cotector treated by tetracycline is inhibited (OD600 nm didn’t increase after incubated under 37℃ in the LB). On the other hand, the OD600 nm of E.Cotector not treated by tetracycline increased. As a result, we proved that the tetracycline will inhibit the growth of E.Cotector.



Figure 2. Our E.Cotector treated by tetracycline (100µg/mL) cannot grow on the LB plate after 37℃ incubated.



Figure 3. The growth of E.Cotector were inhibited by tetracycline.

We put the results of adding different antibiotics in Table 1. Collectively, Tetracycline performed best at killing the bacteria and conserving the fluorescence. From the LB plate result, we observed that the higher the concentration of antibiotics, the less time it would take to fully kill the bacteria. In 4 hours of sterilization, tetracycline ( 100µg/mL) performed better than tetracycline (30µg/mL). Therefore, we chose tetracycline (100µg/mL) as our final safety means.




Table 1. Comparison of different antibiotics affected on the E.Cotector.

How do we know how long it takes for tetracycline to kill all of the E.cotector? After we added tetracycline, we spread the treated bacteria on the LB plate each hour and incubated under 37℃. By counting the number of colonies, we can know the growth situation of E.Cotector. After adding tetracycline for 4 hours, there were no colonies formed (Figure 4.) . So we concluded that it took 4 hours for the tetracycline to kill the bacteria. In other words, our E.Cotector will not grow anywhere after tetracycline treatment for 4 hours.




Figure 4. In the beginning, tetracycline did not play a significant role. After 4 hours though, there were no colonies formed on the LB plate.


After our E.Cotector was treated by tetracycline, we stored the bacteria in the -80℃ refrigerator, and found that the fluorescence was maintained for six days (Figure 5.) .



Figure 5. The fluorescence maintained in the -80℃ refrigerator.



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