Difference between revisions of "Team:NCTU Formosa/Safety"

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 concentration of different antibiotic, 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 to 96 well to detect the fluorescence. There will be more details 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, but in the process, might be because of sulfonamide’s solubility is highly related to the solution’s pH value which causes the solubility to decrease causing the concentration of sulfonamide to distribute unequally. The lower the pH value, the better the solubility of sulfonamide is. This also means, sulfonamide’s 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.

Second step, after added tetracycline, we also extracted bacterial liquid from each sample and added on to LB plate each hour, and cultivate them at 37℃. Once there are bacterial colonies, we determine that the antibiotics didn’t fully kill the bacteria. Experimental results showed that when we add Ampicillin to the bacteria, bacteria could still grow on LB plate as untreated one. On the other hand, bacteria added with Tetracycline won’t grow on the LB plate (Figure 2.).

According to (Figure 3.), we can find that the growth of our E.Cotector treated by tetracycline is inhibited (OD600nm didn’t increase after incubated under 37℃ in the LB). On the other hand, the OD600nm of E.Cotector not treated by tetracycline increased. So we prove 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 result 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 concentration of antibiotics, the less time it takes 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 effected 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 to let tetracycline 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 hour, there were no colony formed on the LB plate.

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

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