IGEM:NYMU/2007/V&V: Difference between revisions

Revision as of 12:45, 20 May 2008

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

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

cell lines
- liver cell (?)
- rat muscle cell (L6)
- mouse adipocyte (3T-3L1) and info. at ATCC
  - 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)
A Ken's progress