NYMU Taipei/Key experiments

From 2007.igem.org

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<h3>System construct</h3>
 +
<ul>
 +
    <li>part extraction</li>
 +
    <li>transformation</li>
 +
    <li>digestion check</li>
 +
    <li>assembly ([http://partsregistry.org/Assembly:Standard_assembly standard assembly] and [http://openwetware.org/wiki/Silver:_BB_Strategy fusion assembly avoidiing frame shift])</li>
 +
</ul>
 +
<h3>Product secretion</h3>
 +
<ul>
 +
    <li>insulin antibody</li>
 +
    <li>IDE antibody</li>
 +
</ul>
 +
<h3>Glucose Sensing Assay</h3>
 +
<ul>
 +
    <li>Objective
 +
    <ul>
 +
        <li>assay for glucose-sensing singaling pathway to verifiy the glucose sensor works or not</li>
 +
    </ul>
 +
    </li>
 +
    <li>Design
 +
    <ul>
 +
        <li>reporting vector: vector with pOmpC + RBS + EYFP (G1 - G7 on box #1)</li>
 +
    </ul>
 +
    <ul>
 +
        <li>factor #1: plasmid with
 +
        <ul>
 +
            <li>empty insertion (as control, bassal level expression)</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>[[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>
 +
        </ul>
 +
        </li>
 +
        <li>factor #2: glucose present or absent in LB medium (total volme 5mL)
 +
        <ul>
 +
            <li>experiment #1
 +
            <ul>
 +
                <li>five tubes for 0, 1%, 3%, 5%, 10% glucose (glucose solution is 20%)</li>
 +
                <li>started at 11:30 PM 9/26</li>
 +
                <li>ended at 13:30 PM 9/27 (14hr duration)</li>
 +
                <li>perform OD test</li>
 +
            </ul>
 +
            </li>
 +
        </ul>
 +
        </li>
 +
    </ul>
 +
    </li>
 +
</ul>
 +
<h3>Circuit Level Assay</h3>
 +
<ul>
 +
    <li>STEP 1: insulin transport by TAT signal peptide induced by glucose
 +
    <ul>
 +
        <li>use insulin antibody to verify the existence of insulin in the medium
 +
        <ul>
 +
            <li>inside the cell</li>
 +
        </ul>
 +
        <ul>
 +
            <li>outside the cell</li>
 +
        </ul>
 +
        </li>
 +
        <li>use Northern blot to verify the existence of CinR and HSL (?)</li>
 +
    </ul>
 +
    </li>
 +
    <li>STEP 2: extract medium from STEP 1 and add into another set of cells which is not induced by glucose
 +
    <ul>
 +
        <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>use <a href="http://www.abcam.com/index.html?datasheet=25970">IDE antibody</a> to verify the existence of IDE</li>
 +
    </ul>
 +
    </li>
 +
</ul>
 +
<h3>Mammalian Cell Assay</h3>
 +
<ul>
 +
    <li>cell lines
 +
    <ul>
 +
        <li>liver cell (?)</li>
 +
    </ul>
 +
    <ul>
 +
        <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>
 +
        <ul>
 +
            <li><a href="http://www-personal.umich.edu/~macdouga/Protocols/Differentiation%20protocol.html">3T-3L1 differentiation protocol</a></li>
 +
        </ul>
 +
        </li>
 +
    </ul>
 +
    </li>
 +
    <li>cluture of cell line</li>
 +
    <li>insulin responsive markers
 +
    <ul>
 +
        <li>insulin level (required insulin antibody)</li>
 +
    </ul>
 +
    <ul>
 +
        <li>glucose level (required glucose detection kit)</li>
 +
    </ul>
 +
    <ul>
 +
        <li>translocation of Glut 4 (required glut4 antibody)</li>
 +
    </ul>
 +
    <ul>
 +
        <li>phosphorylation level of IRS1 (required phospho- insulin receptor substrate-1 antbodies)</li>
 +
    </ul>
 +
    <ul>
 +
        <ul>
 +
            <li>use two different antobodies IRS1 and IRS1(p)</li>
 +
            <li>[http://www.biomedcentral.com/1741-7007/2/23 Acetylation of insulin receptor substrate-1 is permissive for tyrosine phosphorylation, 2004 BMC biology]</li>
 +
        </ul>
 +
    </ul>
 +
    </li>
 +
    <li>[[最新進度|A Ken's progress]]</li>
 +
</ul>
[https://2007.igem.org/NYMU_Taipei/Welcome Back]
[https://2007.igem.org/NYMU_Taipei/Welcome Back]

Revision as of 13:39, 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 <a href="http://www.abcam.com/index.html?datasheet=25970">IDE antibody</a> to verify the existence of IDE

Mammalian Cell Assay

Back

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