NYMU Taipei/Key experiments
From 2007.igem.org
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<li>complete EnvZ insertion (it can be activated by osmotic pressure, calcium)</li> | <li>complete EnvZ insertion (it can be activated by osmotic pressure, 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>[ | + | <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> | ||
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</ul> | </ul> | ||
</li> | </li> | ||
| - | <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/ | + | [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
- experiment #1
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 (?)
- use insulin antibody to verify the existence of insulin in the medium
- 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
