Team:Nankai/Practices
Human Practice: An Amazing Summer！！
As Peter Carr puts it, "Human Practices is the study of how your work affects the world, and how the world affects your work." This summer, Nankai iGEM team worked our best to put the spirits of collaboration and sharing into practice. Not only have we attended many important activities and conferences, but also a new information-sharing platform— iShare has become reality. Let’s see what our team did in this amazing summer!!
1. What is iShare?
During the summer, we ran into many problems when we were working on our experiments. The lack of materials for instance, is one major difficulty that occurred to us. To continue our project, we had to acquire those materials from other laboratories, which involved us into intellectual property issues..
Thanks to the current system of material transformation, we managed to put all the things we needed together. Most of the laboratories would share their resources with us if we sign the material transfer agreement with them. However a small community as Nankai University is, it still took us a long time to find the right materials we needed. We could imagine how difficult if an iGEM team seeks for information about certain materials among the hundreds of previous teams.
4. What did we do?
In order to improve γ-PGA production, we employed two strategies to fine-tune the synthetic pathways and balance the metabolism in the glutamate-independent B. amyloliquefaciens NK-1 strain. Firstly, we constructed a metabolic toggle switch in the NK-1 strain to inhibit the expression of ODHC (2-oxoglutarate dehydrogenase complex) by adding IPTG in the stationary stage and distribute the metabolic flux more frequently to be used for γ-PGA precursor-glutamate synthesis. As scientists had found that the activity of ODHC was rather low when glutamate was highly produced in a Corynebacterium glutamicum strain. Second, to balance the increase of endogenous glutamate production, we optimized the expression level of pgsBCA genes (responsible for γ-PGA synthesis) by replacing its native promoter to seven different strength of promoters. Through these two strategies, we aimed to obtain a γ-PGA production improved mutant strain.Click for more detail.
5. How do we use γ-PGA?
We prepared SOD loaded γ-PGA hydrogel for wound healing. SOD was loaded into hydrogels to scavenge the superoxide anion and γ-PGA was modified with taurine to load more SOD. γ-PGA hydrogel had high water absorption properties delivering the important moist environment. SOD released from the hydrogel maintained high enzyme activity and SOD-γ-PGA hydrogel could scavenge the superoxide anion effectively. In vivo results showed that SOD-γ-PGA hydrogel could promote collagen deposition, epithelialization, and accelerate the healing of moderately exuding wounds. Therefore, SOD-γ-PGA hydrogel would be a good candidate for wound healing applications. Learn more on Pudding Health Kit.
Preparing for LB medium.
Cultured LL3.
In the progress of fermentation.
Our primer.
Ready for doing PCR.
References
1. Ashiuchi, M., Misono, H., 2002. Biochemistry and molecular genetics of poly-γ-glutamate synthesis. Appl. Biochem. Biotechnol. 59, 9–14. 2. Kunioka, M., 1997. Biosynthesis and chemical reactions of poly(amino acid)s from microorganisms. Appl. Microbiol. Biotechnol. 47, 469–475. 3. Shih, I.L., Van, Y.T., 2001. The production of poly(γ-glutamic acid) from microorganism and its various applications. Bioresour. Technol. 79, 207–225. 4. Li, C., 2002. Poly(L-glutamic acid)--anticancer drug conjugates. Adv. Drug Deliver. Rev. 54, 695–713. 5. Liang, H.F., Chen, C.T., Chen, S.C., Kulkarni, A.R., Chiu, Y.L., Chen, M.C., Sung, H.W., 2006. Paclitaxel-loaded poly(γ-glutamic acid)-poly(lactide) nanoparticles as a targeted drug delivery system for the treatment of liver cancer. Biomaterials. 27, 2051–2059. 6. Richard, A., Margaritis, A., 2001. Poly (glutamic acid) for biomedical applications. Crit. Rev. Biotechnol. 21, 219–232. 7. Park, Y.J., Liang, J., Yang, Z., Yang, V.C., 2001. Controlled release of clot-dissolving tissue-type plasmmogen activator from a poly(L-glutamic acid) semi-interpenetrating polymer network hydrogel. J. Control. Release. 74, 243–247. 8. Cao, M.F., Geng, W.T., Liu, L., Song, C.J., Xie, H., Guo, W.B., Jin, Y.H., Wang, S.F., 2011. Glutamic acid independent production of poly-γ-glutamic acid by Bacillus amyloliquefaciens LL3 and cloning of pgsBCA genes. Bioresour. Technol. 102, 4251–4257. 9. Geng, W.T., Cao, M.F., Song, C.J., Xie, H., Liu, L., Yang, C., Feng, J., Zhang, W., Jin, Y.H., Du, Y., Wang, S.F., 2011. Complete genome sequence of Bacillus amyloliquefaciens LL3, which exhibits glutamic acid-independent production of poly-γ-glutamic acid. J. Bacteriol. 193, 3393–3394. 10. Feng, J., Gao, W.X., Gu, Y.Y., Zhang, W., Cao, M.F., Song, C.J., Zhang, P., Sun, M., Yang, C., Wang, S.F., 2014a. Functions of poly-gamma-glutamic acid (γ-PGA) degradation genes in γ-PGA synthesis and cell morphology maintenance. Appl. Microbiol. Biotechnol. 98, 6397–6407. 11. Uy, D., Delaunay S., Germain, P., Engasser, J.M., Goergen, J.L. 2003. Instability of glutamate production by Corynebacterium glutamicum 2262 in continuous culture using the temperature-triggered process. J. Biotech. 104, 173-184.