Difference between revisions of "Part:BBa K245114:Experience"
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+ |    After finishing the design of DenTeeth, we wanted to know whether its inhibition ability can have a function, so we designed the following experiment. Our team decided to use the inhibition zone experiment to confirm that DenTeeth can inhibit other bacteria. Furthermore, we can also compare the difference of inhibition zone diameter to know the strength of inhibition intensity. | ||
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+ |    First, we spread <i>E. coli</i> (DH5α) with pET32a in LB plates with AMP resistance to be our inhibition target. Second, sticking up the filter paper, we dropped the following materials on the filter paper. Finally, we placed LB plates at 37 Celsius degrees for 12 hours. | ||
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+ |    There are ten different materials added to LB plates. Plate A was added with hydrogen peroxide as the positive control group. We could see that Plate A only grew bacteria near the edge. Plates B and C are <i>E. coli</i> (BL21) with pSB1K3 and DenTeeth, respectively. In these two plates, both of which contained K resistance. With other factors unchanged, Plate B is negative control. As we could see, our DenTeeth had an inhibitory effect. In plate D and plate E, are cell lysate of <i>E. coli</i> (BL21) with pSB1K3 and DenTeeth, respectively. In these two plates, both of which contain K resistance. With other factors unchanged, Plate D is negative control. As we could see, the cell lysate of DenTeeth had an inhibitory effect. According to the results of plate A to plate E, we could know whether it is a live DenTeeth or a cell lysate, both of which had the function of inhibiting bacteria. | ||
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+ |    In plates F, G and H, are added with PBS, <i>E. coli</i> (BL21) with pSB1K3 + PBS and DenTeeth + PBS, respectively. To ensure PBS would not influence the inhibition results significantly, we decided to make three plates containing PBS because it would be used on cell lysis. In these three plates, we could observe that only plate H (DenTeeth + PBS) had an inhibitory effect. This result allowed us to know that PBS would not have an obvious influence on our experiment, and can know that DenTeeth still had an inhibitory effect. | ||
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+ |    In plates, I and J, are the LB broth after centrifugation from <i>E. coli</i> (BL21) with pSB1K3 and centrifugation from DenTeeth, respectively. With other factors fixed, we could observe that plate J had an inhibitory effect. This result allowed us to know that antimicrobial peptide LL-37 can be secreted from DenTeeth to the environment. | ||
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[[File:T--NCTU_Formosa--Zone of inhibition.png|500px|thumb|center|'''Figure 7. DenTeeth inhibition zone result in LB plates. ( The materials added in plate were in the following condition, and each total volume is 20μL. (A) 30% hydrogen peroxide, (B) <i>E. coli</i> (BL21) with pSB1K3, (C) DenTeeth, (D) <i>E. coli</i> (BL21) with pSB1K3 (Cell Lysate), (E) DenTeeth (Cell Lysate), (F) PBS, (G) <i>E. coli</i> (BL21) with pSB1K3 + PBS, (H) DenTeeth + PBS, (I) LB broth (Centrifuging <i>E. coli</i> (BL21) with pSB1K3 after liquid culturing ), (J) LB broth (Centrifuging <i>E. coli</i> (BL21) with pSB1K3 after liquid culturing ) ) | [[File:T--NCTU_Formosa--Zone of inhibition.png|500px|thumb|center|'''Figure 7. DenTeeth inhibition zone result in LB plates. ( The materials added in plate were in the following condition, and each total volume is 20μL. (A) 30% hydrogen peroxide, (B) <i>E. coli</i> (BL21) with pSB1K3, (C) DenTeeth, (D) <i>E. coli</i> (BL21) with pSB1K3 (Cell Lysate), (E) DenTeeth (Cell Lysate), (F) PBS, (G) <i>E. coli</i> (BL21) with pSB1K3 + PBS, (H) DenTeeth + PBS, (I) LB broth (Centrifuging <i>E. coli</i> (BL21) with pSB1K3 after liquid culturing ), (J) LB broth (Centrifuging <i>E. coli</i> (BL21) with pSB1K3 after liquid culturing ) ) | ||
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− |    | + |    We repeated the DenTeeth inhibition functional test three times each. In figure 5, this bar graph indicates the three times average of inhibition zone diameters in different materials added to LB plates. This result shows that DenTeeth can secrete LL-37 to inhibit other bacteria. |
[[File:T--NCTU_Formosa--Zone of inhibition2.png|500px|thumb|center|'''Figure 8. The Bar Chart of DenTeeth inhibition Functional Test. | [[File:T--NCTU_Formosa--Zone of inhibition2.png|500px|thumb|center|'''Figure 8. The Bar Chart of DenTeeth inhibition Functional Test. |
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