Contribution
Besides our team, a lot of previous iGEM teams aimed to relieve the symptom of diabetes, such as 2019 iGEM team_Bilkent-UNAMBG trying to create a probiotic E. coli which we can be orally administrable, 2019 iGEM team_BSC_United’s project “MINILOSS” (MIcrofluidic organ chIp for blood glucose stabilization) which commits to an innovative, painless, bio-based approach for diabetic treatment, and 2020 iGEM team_ECNUAS created a platform for screening GR antagonists which might alleviate T2DM.
We aimed to relieve burdens caused by diabetes, such as economic stress, and its serious side effects to bring well-being to people with diabetes.
The transformation of PQQ gene in Bacillus subtilis natto indeed synthesized PQQ and was capable of helping cells to recover or to defend against oxidative stress. However, we needed further experiments to quantify PQQ.
We hoped the parts BBa_K4358011 and BBa_K4358012, which we defined from BBa_K3830012 would help further research by future iGEM teams about diabetes, PQQ, or wound healing.
Figure 2. The growth curve of B. subtilis natto wild type and EP2, B. subtilis natto with BBa_K3830012. We compared the growth condition with the bacteria used by 2021 NCHU_Taichung.
It showed that EP2 and EP2 kanamycin had more growth ability than RM125, RM125PQQ and natto. It has proved that, by this experiment, PQQ could indeed bring a positive effect on the growth of B. subtilis.
Improvement
pqqA is the precursor of PQQ, a short peptide with twenty-six amino acids that contain glutamate and tyrosine. pqqD and pqqE help generate the covalent bond within pqqA. Then pqqB will catalyze the generation of AHQQ, which acts as the substrate in the final oxidative steps and in the rate-determining of the pathway. Then AHQQ would be catalyzed to become PQQ by pqqC.
We created a new part, BBa_K4358011, which was designed based on the part, BBa_3830012 with Pxyl, xylR and xylR-binding sites. The pqqA and pqqB are crucial for PQQ synthesis.
Based on the document that 2021 NCHU_Taichung provided, it has been known that pqqA is the precursor of PQQ. In 2022, we, NCHU_Taichung, plan to amplify PQQ production by doubling pqqA in our new plasmid BBa_4358013.
Different from BBa_K3830012, BBa_4358013 had more potential to synthesize pqqA. And BBa_4358013 may improve the efficiency of PQQ production, and make the PQQ produced by modified bacteria much more likely to be applied to commercial use.
To magnify the expression of pqqA and pqqB we designed a xylose operon to make energy distribution controllable. To amplify the biosynthesis of pqqA and pqqB we may shut down the energy distribution to produce pqqC, pqqD, and pqqE to enhance the energy distribution of pqqA and pqqB biosynthesis indirectly.
Xylose, which is an inducer binding the repressor “XylR ”, would not be added to the culture medium; on the contrary, to synthesize PqqC, PqqD, and PqqE, the addition of xylose would be required for pqqC, pqqD and pqqE to express.
In conclusion, we made Bacillus subtilis natto with BBa_4358011 more effective for the biosynthesis of PqqA and PqqB and it could be controlled more precisely.
Figure 2. Improvement part: composite part BBa_4358011.
Zhu, W., & Klinman, J. P. (2020). Biogenesis of the peptide-derived redox cofactor pyrroloquinoline quinone. Current opinion in chemical biology, 59, 93–103.
https://doi.org/10.1016/j.cbpa.2020.05.001
