Team:Think Edu China/Engineering


Step 1: Design the mechanism of degradation

After established a thourough understanding of our project direction, which is the degradation of antibiotics, we selected a specific enzyme named laccase 6 as the major player for the process. According to research that we have done, the laccase enzyme was previously used in enzymatic membrane reactor (EMR) as a mean to remove the antibiotics. Therefore, we cut out a specific gene fragment laccase 6 to be inserted into our constructed bacterial strain.

Step 2: Construct the whole cell catalyst

After designed our engineered plasmid, in order to determine whether or not the gene lacc 6 was successfully anchored on the extra cellular membrane of the bacteria strain, the study utilized overlap PCR to connect gene gfp and the end of INP-N-Lacc6. Using fluorescence microscopy, the recombinant strain ECN-ILG observed to emit green fluorescence, while the control strain ECN-PSB18A cannot. Thus, came to the conclusion that Lacc6 gene fragment was successfully anchored on the surface of EcN cells surface.

Step 3: Test the sensitivity of bacterial strain toward antibiotics

In order to ensure that the engineered bacterial strain can be obtain by the antibiotic resistant screening, the sensitivity of the wild strain of EcN toward several types of antibiotics should be determined. In order to test the sensitivity of the bacterial strain, we inoculate the EcN strain into test tubes that each contains Chloramphenicol (30 μg/ mL), Ampicillin (50 μg/ mL) and Kanamycin (50 μg/ mL). Surprisingly, after 12 hours of incubation, if the wild-type EcNs cannot survive in the presence of the above antibiotics that is used for cloning screening, the strain can then be used as host bacteria serves to express the protein.

Step 4: Advancement and redesigned the EcN-IL

After we have found out that the EcN cannot really survive amount the antibiotics, we were thinking of a way to increase the efficiency of degradation. Instead of directly apply laccase to antibiotics, we construct a new system using the cell surface display technology. Sulfonomide antibiotics is known for not kill bacteria, but it interferes with the ability of bacteria to grow and multiply. Thus, we learn the Nissle 1916 won't be killed by the Sulfonomide. For the advancement of the project, we construct the whole cell catalyst that utilize INP-N protein to anchor the laccase to the extracellular membrane of EcN to form EcN-IL.

Step 5: Test analysis of the sensitivity of the constructed EcN-IL

After EcN-IL was created , we tested its rate of degradation in vitro. We used ABTS as substrates and the tested laccase activity of whole cell catalyst was 1.99 ± 087 U/ cell dry weight when the absorbance value was 420nm (Fig. 3-1). while the enzyme activity was not detected in ECN - p18a and wild-type EcN.

Step 6: Secondary design of the optimal operation conditions

After find out the EcN-IL has comparable effect in degradation of Sulfonomide antibiotics, we attempt to search for the best condition for the enzymes to process. We specifically investigated the effects of pH level and temperature on the degradation rate of sulfadiazine by the engineering strains. As the result shows in Figure 3-2, when the pH level is in the range of 4~7, the degradation rate of ECN on SDZ gradually increases, and reaches the maximum when pH level equals to 7.0. With the gradual increase of pH, the degradation efficiency began to decreases gradually. Moreover, Fig. 3-2 also shows that the degradation rate of SDZ increases gradually with the increase of temperature, and reaches highest around 40 ℃. Therefore, the optimum pH level is 7.0 with temperature of 40 ℃.