Team:NCTU Formosa/Wet Lab/Safety

Our system provides a nice Biosimulator, bacterioicn, which shows the antimicrobial activity against specific strains. Since we plan to utilize our product into reality, we have to make sure that these peptides will not cause any safety problems and our modified bacteria will not be spread into the environment.

Bacteriocins are natural peptides and degrade quickly in the environment. Therefore, the most important thing we need to confirm is the modified E.coli. Thus, we cut into different aspects to ensure the safety of our product: the training of the experimenter, the level and the rule of the laboratory, and the safety of our final products using in the environment.

Safety Training and Laboratory

Because the modified E.coli may be harmful when released to the environment, our experiments are done in the BSL2 laboratory. All of our team members receive several training courses and pass exams offered by Laboratory Management System before entering the laboratory. Each member must wear the lab coat, trousers, gloves, surgical masks, and shoes when carrying out experiments. Before and after the experiments, we have to use ethanol to clean the gloves and the experiment table. Everybody strictly follow the experimental procedure and never carry anything out of the lab.

Confirm the Safety of Bio-stimulator

- High-temperature Sterilization Target on Modified E.coli ER2566 -

In the future, we are going to spray our Bio-stimulator into the environment. To make sure whether the bacteria contain anti-microbial peptide will not exist in the final product, we design the processing standards in the laboratory.

Bacteriocins are usually heat stable, we use high-temperature sterilization to double make sure our peptide solution does not contain any living E. coli. However, peptides may degrades after long time sterilization. To find out the best fitted time for sterilization, we boiled our bacteriocins for 0, 15, 30, and 45 minutes, and put them on LB Agar plate and cultured it at 37℃ for 16 hours.

These are the result of the plates, we can easily observe that the bacteria exists only in the sample that is not boiled. After fifteen minutes of sterilization, there are no alive bacterias exist.

Figure 1: LB Agar plate of boiling test to Enterocin 96+intein+CBD(35.9kDa) and Enteroicin B+intein+CBD(35.5kDa)

Figure 2: LB Agar plate of boiling test to Bovicin HJ50+intein +CBD(34.25kDa) and Durancin +intein+CBD(35.3kDa)

Figure 3: LB Agar plate of boiling test to Lacticin+intein+CBD(33.9kDa) and Leucocyclicin Q

We also run SDS-PAGE to check the degradation of bacterioicins. The result shows that the peptide doesn’t degrade after boiling in 45 minutes.

Figure 4: SDS-PAGE gel and the concentrations of boiling test to bacteriocins.

(A) Enterocin 96+intein+CBD(35.9kDa) (B) Enteroicin B+intein+CBD(35.5kDa)

Figure 5: SDS-PAGE gel and the concentrations of boiling test to bacteriocins.

(C)Bovicin HJ50+intein +CBD(34.25kDa) (D) Durancin +intein+CBD(35.3kDa)

Figure 6: SDS-PAGE gel and the concentrations of boiling test to bacteriocins.

(E) Lacticin+intein+CBD(33.9kDa)

Figure 7: SDS-PAGE gel and the concentrations of boiling test to bacteriocins.

(F) Leucocyclicin Q+intein+CBD(34.4kDa)