Team:NWU-CHINA-B/Contribution

NWU-CHINA-B

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  • Overview


    1. The handbook of Yeast


      2. Together with CPU_CHINA, Jiangnan_China, UESTC-China, UM_China, HUST-China, HIZJU-China and NWU-CHINA-B, we compiled the yeast handbook, which contributed to other teams' reference to ginsenoside knowledge. The book records some new methods, experimental summary and experience of our yeast experiment, and records some common mistakes in yeast experiment.



    2. Original case


      3. For the contribution, we completed the experimental characterization of the previous parts (BBa_K3196099 and BBa_K392001) and added the data of them to the corresponding BioBricks. All of these may be helpful to other teams. We hope it will make some contribution to the iGEM community.







    3. Contribution



      4. ① α-factor:( BBa_KK3196009)



      α-factor signal peptide is a leading peptide of mating factor 1 (MF1) N-terminus secreted by yeast α cells. It consists of 86 amino acid residues, including pre-sequence and pro-region sequences. Studies have shown that α-factor signal peptides play an important role in the secretion of exogenous proteins in Pichia pastoris, and the three N-glycosylation of pro-region is important for the secretion of peptides.

      We improved the structure and function of α-factor and explained the processing of α-factor-guided exogenous protein in the secretion pathway, which helped us to deepen our understanding of α-factor. Meanwhile, we summarized the endogenous signal peptide expressed by exogenous protein in Pichia pastoris. This will facilitate the expression and optimization of pichia pastoris α-factor endogenous signal peptide in the future.


      We compared the difference in the expression of exogenous protein guided by S.cerevisiae α-factor signal peptide with N-glycosylation modification and without N-glycosylation modification. The linearized recombinant plasmid pPIC9KS. The recombinant GS115S. Ce-N23/57/67Q-EGFP was obtained by electric transfer, and cultured together with GS115S. Ce-EGFP for 24h BMGY. Under the same conditions (OD=1, volume: 5ml), the supernatants were harvested for 72h after methanol induction, and western-blot analysis was performed. The results showed that EGFP secretion was decreased by 50.3% after the mutation of N to Q at three potential glycosylation sites of S.cerevisiae α-factor signaling peptide.


      The results showed that S.cerevisiae α-factor signal peptide was modified by N-glycosylation when expressed in Pichia pastoris, and N-glycosylation of S.cerevisiae α-factor signal peptide contributed to the improvement of exogenous protein production.




      ② Yeast ENO2 promoter：(BBa_K392001)



      Through data collection, we know that the ENO2 promoter has the function of driving the high-level expression of differentiated neuronal genes in vertebrates, so the study of ENO2 promoter plays a crucial role in the study of neurodegenerative diseases in vertebrates. Zebrafish, as a vertebrate, has been widely used in the study of human diseases due to its high genetic similarity with human 87 percent such as: valuable insights into disease pathogenesis, or leads to new therapeutic approaches, could be provided by high-content small molecule screens, genetic suppressor screens, observation of disease progression in real time in vivo, using fluorescent reporters to label cell populations of interest, and rapid hypothesis-testing experiments in statistically robust samples of larvae .

      In the study of transgenic zebrafish, it is clearly recognized that the high expression of ENO2 can prevent neuronal degeneration by constructing neurodegeneration model, which makes an important contribution to the research on neurodegenerative diseases in the world today. The ENO2 promoter will enable us to test the hypothesis that biochemical and histological changes represent of human diseases may be provoked in susceptible neuronal populations by expression of mutant transgenes.

      The 12 kb ENO2 promoter fragment is sufficiently small that insertion of transgene sequences into the plasmid construct is straightforward; the resulting plasmid can be grown at a high copy number in Escherichia coli and DNA preparations made with ease. In addition, the relatively compact nature of the construct allows for efficient transgenesis using the I-Sce1 technique.

      This figure in the literature confirm the specificity of the ENO2 expression pattern demonstrated and show that the widespread hybridization pattern of the ENO2 cRNA probe to brain sections is attributable to pan-neuronal expression of ENO2 rather than non-specific probe hybridization.



      This figure in the literature suggest that the ENO2 promoter drives high-level transgene expression in adult zebrafish brain.








      Reference:

      ①Zou Chenwei, Huang Yide. Research progress of pichia pastoris signal peptides secreted by exogenous proteins [J]. Biotechnology letters,2020,31(06):781-785.

      ②Wu J W. Effect of α-factor signal peptide and n-glycosylation modification on exogenous protein secretion in Pichia pastoris [D]. Fujian Normal University,2019.

      ③Qing Bai, Jessica A. Garver, Neil A. Hukriede, Edward A. Burton, Generation of a transgenic zebrafish model of Tauopathy using a novel promoter element derived from the zebrafisheno2gene ,Nucleic Acids Research, Volume 35, Issue 19, 1 October 2007, Pages 6501–6516