From 2007.igem.org

Contents 1 What we need in our project 2 Beginning of Story 3 Action plan 4 Make for practical use

What we need in our project

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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

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Action plan

• Chih-Hsien Yang had concluded again in meeting record 2007-0716
• sensing glucose
  • glucose-responsive promoter
    • two component system for glucose sensing in E.coli
    • iGEM 2007 parts and basic system configuration
      • OmpR binding site
        • Part:BBa_R0082 (15P, plate 1)
        • Part:BBa_R0083 (17H, plate 1)
        • Part:BBa_R0084 (11H, plate 1)
      • EnvZ and RcsC fusion construct
        • tar-EnvZ 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
      • 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
      • g1005043C07: MGC Human Verified Full-length cDNA Collection (IRAU), IMAGE id=3950204 (from BCRC, 4,000 NTD)
      • Construct details
      • 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
      • 生理所阮琪昌老師: they don't have
      • Library Resources Services at Gene Expression Analysis Center, VYMGC
        • 訂購流程  and find genes by MGC finder
        • 8/14 送出, 8/17 預定送來
    • foreighn sources
    • DNA synthesis or gene cloning
    • iGEM registry search (iGEM part registry and iGEM 2007 kit)
      • A (alpha) chain of human insulin
      • B (beta) chain of human insulin
      • full length of human insulin gene
    • insulin antibody
• secreting insulin
  • insulin transportation
    • by signal peptide for protein sorting
      • SRP: 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: Inhibition of Insulin Receptor Activation by Insulin-like Growth Factor Binding Proteins, 1997 JBC (posted by 迪璿)
    • inhibitory anti-sense RNA for insulin mRNA (posted by Chih-Hsien)
    • [Comparison of insulin regulatory models]
• 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

 

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Make for practical use

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

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