From 2007.igem.org

Contents 1 System construct 2 Product secretion 3 Glucose Sensing Assay 4 Circuit Level Assay 5 Mammalian Cell Assay

System construct

• part extraction
• transformation
• digestion check
• assembly ([http://partsregistry.org/Assembly:Standard_assembly standard assembly] and [http://openwetware.org/wiki/Silver:_BB_Strategy fusion assembly avoidiing frame shift])

Product secretion

• insulin antibody
• IDE antibody

Glucose Sensing Assay

• Objective
  • assay for glucose-sensing singaling pathway to verifiy the glucose sensor works or not
• Design
  • reporting vector: vector with pOmpC + RBS + EYFP (G1 - G7 on box #1)
  • factor #1: plasmid with
    • empty insertion (as control, bassal level expression)
    • complete EnvZ insertion (it can be activated by osmotic pressure,  calcium)
    • tar-EnvZ insertion (it can be activated by asparate)
    • RcsC-EnvZ insertion (it can be activated by glucose ideally, RcsF is an essential component)
    • Dr. Chang said: E.coli can response to glucose and activate OmpR directly
  • factor #2: glucose present or absent in LB medium (total volme 5mL)
    • experiment #1
      • five tubes for 0, 1%, 3%, 5%, 10% glucose (glucose solution is 20%)
      • started at 11:30 PM 9/26
      • ended at 13:30 PM 9/27 (14hr duration)
      • perform OD test

Circuit Level Assay

• STEP 1: insulin transport by TAT signal peptide induced by glucose
  • use insulin antibody to verify the existence of insulin in the medium
    • inside the cell
    • outside the cell
  • use Northern blot to verify the existence of CinR and HSL (?)
• STEP 2: extract medium from STEP 1 and add into another set of cells which is not induced by glucose
  • expect the medium in STEP 1 has CinR and HSL
  • without glocuse and with CinR and HSL, system 2 can be induced by CinR+HSL complex and produce IDE
  • use [http://www.abcam.com/index.html?datasheet=25970 IDE antibody] to verify the existence of IDE

Mammalian Cell Assay

• cell lines
  • liver cell (?)
  • rat muscle cell (L6)
  • mouse adipocyte (3T-3L1) and [http://www.atcc.org/common/catalog/numSearch/numResults.cfm?atccNum=CL-173 info. at ATCC]
    • [http://www-personal.umich.edu/~macdouga/Protocols/Differentiation%20protocol.html 3T-3L1 differentiation protocol]
• cluture of cell line
• insulin responsive markers
  • insulin level (required insulin antibody)
  • glucose level (required glucose detection kit)
  • translocation of Glut 4 (required glut4 antibody)
  • phosphorylation level of IRS1 (required phospho- insulin receptor substrate-1 antbodies)
  • use two different antobodies IRS1 and IRS1(p)
  • [http://www.biomedcentral.com/1741-7007/2/23 Acetylation of insulin receptor substrate-1 is permissive for tyrosine phosphorylation, 2004 BMC biology]
• A Ken's progress

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