Bacillus subtilis natto (B. subtilis natto) was isolated from commercially available natto. After sending 16s rDNA PCR sample to Genomics company for sequencing and comparing with the database of NCBI, the result showed that it was 100% similar to Bacillus subtilis natto. We extracted the PQQ plasmid, pET28a, from Bacillus subtilis derived from iGEM 2021 team NCHU-Taichung and did electrophoresis to ensure it was the correct PQQ plasmid with the same base pairs as a pure plasmid. Next, electroporation was utilized to transfer the plasmid into B. subtilis natto. The new engineering bacterium is called EP2. The plasmid extracted from EP2 processed DNA sequencing by electrophoresis with the original plasmid (iGEM 2021 team), and also amplified by the significant primer to check again the EP2 amplified fragments as the same as the amplified fragments come from the original plasmid. Thus far, we confirmed the success of our electroporation and the establishment of an industrial factory, EP2. Afterward, in the qualitative analysis, we measured the growth curve of EP2 and B. subtilis natto (Wild type). The result illustrated that EP2 entered the stationary phase faster than the Wild type. And in the quantitative part, a PQQ separation and purification were found. That allowed the quantitative analysis of PQQ, and further, measured the efficiency of PQQ secretion out of the EP2 membrane.

Figure1. The positive control and the PQQ plasmid from B. subtilis natto PQQ are on the same molecular size.

In the phenotype illustration, the immortalized HaCaT keratinocyte cell was chosen as a platform to carry out the assay of wound healing. We simulate the diabetic wound by treating lots of glucose and insulin to induce cells. Next, the pure PQQ, natto extract, and mixture (pure PQQ mixed with natto extract) were added into the cell. The result shows that no matter what kind of additions we used, the effect of wound healing is much better than the control (no addition). Conclusively, we created an engineering bacterium, Bacillus subtilis natto PQQ (EP2), and with its products, a kind of novel dressing to treat diabetic wounds is in progress.

The growth curve of Ep2(transformed with PQQ plasmid ) and B. subtilis wild type.

Wound healing assay of diabetic-like induced HaCaT cell in control, Natto (natto extraction 50 μg/mL) , PQQ (50 nM) and Mix (natto extraction 50 μg/mL with PQQ 50 nM).

Besides, committed to being scientific and rational, we interview the doctor from the Department of Infectious Diseases and data research, Dr. Chen Changhua. He provided us with lots of professional advices, especially checking humanity and how to fit social needs. After the meet-up, all information would inspire and motivate us to design a new dressing product suitable for the diabetic wound.

Figure 4: Interviewed a the doctor from the Department of Infectious Diseases, Dr. Chen Changhua.


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    Retrieved from https://unstats.un.org/sdgs/report/2021/goal-03/
  2. United Nations. (2022). SDG Goals - United Nations Statistics Division.
    Retrieved from https://unstats.un.org/sdgs/report/2021/goal-07/