NYMU Taipei/Key experiments

From 2007.igem.org

(Difference between revisions)
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         <li>expect the medium in STEP 1 has CinR and HSL</li>
         <li>expect the medium in STEP 1 has CinR and HSL</li>
         <li>without glocuse and with CinR and HSL, system 2 can be induced by CinR+HSL complex and produce IDE</li>
         <li>without glocuse and with CinR and HSL, system 2 can be induced by CinR+HSL complex and produce IDE</li>
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         <li>use <a href="http://www.abcam.com/index.html?datasheet=25970">IDE antibody</a> to verify the existence of IDE</li>
+
         <li>use [http://www.abcam.com/index.html?datasheet=25970 IDE antibody] to verify the existence of IDE</li>
     </ul>
     </ul>
     </li>
     </li>
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     <ul>
     <ul>
         <li>rat muscle cell (L6)</li>
         <li>rat muscle cell (L6)</li>
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         <li>mouse adipocyte (3T-3L1) and <a href="http://www.atcc.org/common/catalog/numSearch/numResults.cfm?atccNum=CL-173">info. at ATCC</a>
+
         <li>mouse adipocyte (3T-3L1) and [http://www.atcc.org/common/catalog/numSearch/numResults.cfm?atccNum=CL-173 info. at ATCC]
         <ul>
         <ul>
-
             <li><a href="http://www-personal.umich.edu/~macdouga/Protocols/Differentiation%20protocol.html">3T-3L1 differentiation protocol</a></li>
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             <li>[http://www-personal.umich.edu/~macdouga/Protocols/Differentiation%20protocol.html 3T-3L1 differentiation protocol]</li>
         </ul>
         </ul>
         </li>
         </li>

Revision as of 14:45, 18 October 2007

Contents

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

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