NYMU Taipei/Key experiments

From 2007.igem.org


System construct

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 IDE antibody to verify the existence of IDE

Mammalian Cell Assay