NYMU Taipei/Materials

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Contents

What we need in our project

Beginning of Story

  • De-Ming Lin had concluded what a insulin-secretory cell should be from interature review
    • Cells engineered for insulin expression should contain
      • Proteases for insuline or proinsulin or express two chains (A, B) separately (activate insulin)
      • glucose-sensing system (sense glucose)
      • secretory granules that can release insulin promptly by exocytosis in response to extracellular glucose levels

Action plan

  • Chih-Hsien Yang had concluded again in meeting record [http://igem.ym.edu.tw/private_2007/index.php/Regulated_insulin_serection_Team_Meeting_2007/07/16 2007-0716]
  • sensing glucose
    • glucose-responsive promoter
      • two component system for glucose sensing in E.coli
      • iGEM 2007 parts and [http://igem.ym.edu.tw/private_2007/index.php/Draft_of_Project_Description basic system configuration]
        • OmpR binding site
          • [http://partsregistry.org/Part:BBa_R0082 Part:BBa_R0082] (15P, plate 1)
          • [http://partsregistry.org/Part:BBa_R0083 Part:BBa_R0083] (17H, plate 1)
          • [http://partsregistry.org/Part:BBa_R0084 Part:BBa_R0084] (11H, plate 1)
        • EnvZ and RcsC fusion construct
          • [http://partsregistry.org/Part:BBa_C0082 tar-EnvZ] [http://partsregistry.org/Part:BBa_C0082 Part:BBa_R0082]
      • lac operon in E.coli
      • native insulin promoter in beta cell of eukaryotic system
        • native beta cell can response glucose to secret insulin
        • [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WG0-4NR188R-2&_user=10&_coverDate=05%2F13%2F2007&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=ee060f4907f3e248cbb91d08b477702a Regulation of insulin gene expression and insulin production in Nile tilapia (Oreochromis niloticus), 2007 General and Comparative Endocrinology]
  • producing insulin
    • insulin included plasmid from
      • domestic sources
        • [http://strain.bcrc.firdi.org.tw/BSAS/controller?event=SEARCH&bcrc_no=g1005043C07&type_id=5&keyword=insulin g1005043C07: MGC Human Verified Full-length cDNA Collection (IRAU), IMAGE id=3950204] (from [http://www.bcrc.firdi.org.tw/wwwbcrc/main.jsp BCRC], 4,000 NTD)
        • Construct details
        • [http://www.dls.ym.edu.tw/t11.htm 生科系蔡亭芬老師] 可提供老鼠 (BL_4) 胰臟組織做 RT-PCR 取 insulin clone (連絡日期 8/3, PM 4:28)
          • 8/6 蔡老師博士班學生吳家瑜 (分機 5529) 會幫忙抽好 RNA, 需要訂購 primer
          • 8/7 拿到RNA, 已存入 -80 度冰箱
        • [http://www.ym.edu.tw/phy/persons/juancc.htm 生理所阮琪昌老師]: they don't have
        • [http://genome.ym.edu.tw/libres/ Library Resources Services at Gene Expression Analysis Center, VYMGC]
          • [http://genome.ym.edu.tw/libres/Ordering%20procedure.htm 訂購流程]  and find genes by [http://www.geneservice.co.uk/products/tools/MGC_Finder.jsp MGC finder]
          • 8/14 送出, 8/17 預定送來
      • foreighn sources
      • DNA synthesis or gene cloning
      • [http://partsregistry.org/partsdb/search.cgi iGEM registry search] ([http://partsregistry.org/Part_Types iGEM part registry] and [http://partsregistry.org/Help:IGEM_07_Parts_Kit iGEM 2007 kit])
        • [http://partsregistry.org/Part:BBa_S03451 A (alpha) chain of human insulin]
        • [http://partsregistry.org/Part:BBa_S03449 B (beta) chain of human insulin]
        • [http://partsregistry.org/Part:BBa_I10075 full length of human insulin gene]
      • [http://www.genwaybio.com/product_info.php?products_id=71169 insulin antibody]
  • secreting insulin
    • insulin transportation
      • by signal peptide for protein sorting
        • SRP: [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=8521806 Early events in preprotein recognition in E.coli: interaction of SRP and trigger factor with nascent polypeptides, 1995 EMBO]
      • by vesicle for material transport in eukaryotic cell
  • activating insulin
    • Proteases (convertase PC2 and PC1/3) for insuline or proinsulin or
    • express two chains (A, B) separately (without chain C)
  • regulating insulin
    • To balance the overexpressed insulin under low glucose supplement
      • insulin protein inhibitor IGFBP7: [http://www.jbc.org/cgi/content/abstract/272/49/30729 Inhibition of Insulin Receptor Activation by Insulin-like Growth Factor Binding Proteins, 1997 JBC] (posted by 迪璿)
  • diabetic mammlian cell line
    • domestic sources
    • foreighn sources

 

       RNA Switch Biosensor 

  1. Influenza virus like particle (VLP)-get from professor Ho, Mei-Shang (Academia Sinica, IBMS )
  2. iNOS promoter and iNOS gene construct-get from professor Lin, Wan-Wan (NTU)
  3. pTREX -GFP construct-get from professor Stephen Elledge (Harvard University)
  4. Cytochrome c- from VYMGC
  5. Antiswitch, TetR, pTet-get from iGEM

 

Make for practical use

  • control the population of E.coli-based insulin secretory cell
  • prevent the immune response

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