Team:NCTU Formosa/Safety
This year Apollo designed a product mainly to detect antigens. Our users include scientific researchers, clinical scientist and so on. We believe these people all have the expertise, but we would like to once again remind everyone that our product contains E. coli. Therefore when applying our product, basic protective gear (lab coat, gloves and so on) are needed. Objects that come into contact with our product (for example: tips) need to be sterilized and thrown away, in case of non-natural gene outflow. We’ll hand 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 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.
Paraformaldehyde
At first, we treated our E.Cotector, displayed scFv(anti-EGFR) and green fluorescence protein(GFP), by paraformaldehyde. Paraformaldehyde is a chemical substance that is toxic for organism. It can fix the cell structure and all of the proteins.
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 [3] [4].
Result
We mixed the antibiotic and bacteria together. Then we observed the growth and the fluorescence of bacteria. There will be more details in the protocol. The category and testing concentration of antibiotic are bellowed.
In the experiment where we add Tetracycline and Ampicillin, we discovered the more antibiotics we added the less the fluorescence(Figure 1). Tetracycline reduces the fluorescence at a higher rate than Ampicillin. But when we cultivate the bacteria on LB plate, we noticed that the whether we add Ampicillin or not, the bacteria still grows on the plate. On the other hand, bacteria added with Tetracycline won’t grow on the LB plate(Figure 2).
Figure 1. Impacted by ampicillin and tetracycline,there were the expression of fluorescence during 12 hours.
Figure 2. The result of incubated the E.Cotector on the LB plate treated by ampicillin or tetracycline.
In the next experiment where we add sulfonamide, we observed that the fluorescence is more or less the same amount as the original bacteria(Figure 3). Correspondingly, we also cultivated the cell on LB plate. We discovered the inhibition of growth doesn’t steadily change with the concentration of Sulfonamide. Some bacteria still grow and some don’t(Figure 4).
Figure 3. Impacted by sulfulnamide,there were the expression of fluorescence during 12 hours.
Figure 4. The result of incubated the E.Cotector on the LB plate treated by sulfonamide.
Conclusion
From the experiment, we learned that different antibiotics have different amounts of fluorescence conservation. The conservation effect: Ampicillin>Sulfonamide>Tetracycline. The conservation amounts are all sufficient for detection. Furthermore, the growth condition of our bacteria treated by different antibiotics also varies. The inhibition effect: Tetracycline>Sulfonamide>Ampicillin. Among the three, Sulfonamide’s effect is unstable. 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. We put the result of adding different antibiotics in Table 1. Lastly, since collectively Tetracycline performed best at killing the bacteria and conserving the fluorescence, we chose tetracycline to continue our safety.
Table 1. Comparison of different antibiotics effected on the E.Cotector.
Since we chose tetracycline as our main focus, we need to test how much concentration of tetracycline can achieve the best sterilization effect. Furthermore, we want to learn how long does it take to let the antibiotic work efficiently so that it cannot grow on the LB plate. We add different concentration of tetracycline to our E. cotector and compare it with E. cotector without tetracycline. After adding the antibiotic, we extracted appropriate amount of bacterial liquid from each sample and added to 96 well to detect the fluorescence. In the mean time, we also extracted 1 uL of bacterial liquid from each sample and added on to LB plate. We repeat this step every hour and continue for 7 hours. Moreover, we cultivate the LB plate long enough to make sure all the living bacteria will grow. We observe the best concentration and best reaction time for tetracycline to effectively kill the bacteria.
NCTU_Formosa APOllO
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