Difference between revisions of "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.
According to the result, we found that paraformaldehyde-treated E.coli can grow in the LB broth at 37℃ incubated.

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(Fig.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(Fig.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(Fig.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(Fig.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.