NYMU Taipei/Key experiments

From 2007.igem.org

(Difference between revisions)
 
(3 intermediate revisions not shown)
Line 28: Line 28:
             <li>complete EnvZ insertion (it can be activated by osmotic pressure,&nbsp; calcium)</li>
             <li>complete EnvZ insertion (it can be activated by osmotic pressure,&nbsp; calcium)</li>
             <li>tar-EnvZ insertion (it can be activated by asparate)</li>
             <li>tar-EnvZ insertion (it can be activated by asparate)</li>
-
             <li>[[RcsC-EnvZ]] insertion (it can be activated by glucose ideally, RcsF is an essential component)</li>
+
             <li>[https://2007.igem.org/NYMU_Taipei/Key_experiments/RcsC-EnvZ RcsC-EnvZ] insertion (it can be activated by glucose ideally, RcsF is an essential component)</li>
             <li>Dr. Chang said: E.coli can response to glucose and activate OmpR directly</li>
             <li>Dr. Chang said: E.coli can response to glucose and activate OmpR directly</li>
         </ul>
         </ul>
Line 66: Line 66:
         <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>
-
         <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>
Line 78: Line 78:
     <ul>
     <ul>
         <li>rat muscle cell (L6)</li>
         <li>rat muscle cell (L6)</li>
-
         <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>
+
             <li>[http://www-personal.umich.edu/~macdouga/Protocols/Differentiation%20protocol.html 3T-3L1 differentiation protocol]</li>
         </ul>
         </ul>
         </li>
         </li>
Line 106: Line 106:
     </ul>
     </ul>
     </li>
     </li>
-
     <li>[[最新進度|A Ken's progress]]</li>
+
     <li>[https://2007.igem.org/NYMU_Taipei/Key_experiments/A_Ken's_progress A Ken's progress]</li>
</ul>
</ul>
-
[https://2007.igem.org/NYMU_Taipei/Welcome Back]
+
[https://2007.igem.org/Taipei Back]

Latest revision as of 16:50, 25 October 2007

Contents

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

Back

Retrieved from "http://2007.igem.org/wiki/index.php/NYMU_Taipei/Key_experiments"