Team:NCTU Formosa/Wet Lab/Safety

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     Our system provides a nice bio-stimulator, 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 BL21 Rosetta-gami

     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 results 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 bacteria exist.

Figure 1: Confirm E. coli BL21 Rosetta-gami doesn’t grow in LB Agar plate after high-temperature sterilization.
Samples are Enterocin 96 + intein + CBD(35.9 kDa) and Enteroicin B + intein + CBD(35.5 kDa)

Figure 2: Confirm E. coli BL21 Rosetta-gami doesn’t grow in LB Agar plate after high-temperature sterilization.
Samples are Bovicin HJ50 + intein + CBD(34.25 kDa) and Durancin + intein + CBD(35.3 kDa).

Figure 3: Confirm E. coli BL21 Rosetta-gami doesn’t grow in LB Agar plate after high-temperature sterilization.
Samples are Lacticin + intein + CBD(33.9 kDa) and Leucocyclicin Q + intein + CBD(34.4 kDa).

     We also ran SDS-PAGE to check the degradation of bacteriocins. The result showed that the peptide didn’t degrade after boiling in 45 minutes. We chose Enterocin 96 and Enterocin B as the representatives. From this, we deduced similar results from the other bacteriocins.

Figure 4: Determine the stability of bacteriocins after high-temperature sterilization. The experimental samples are Enterocin 96+intein + CBD (35.9 kDa) and Enterocin B+ intein + CBD (35.5 kDa).
M: Protein Ladder 5–245 kDa; A: Sample without high-temperature sterilization; B: Sample sterilize at 100℃ for 15 mins; C: Sample sterilize at 100℃ for 30 mins; D: Sample sterilize at 100℃ for 45 mins.