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Team:SJTU-BioX-Shanghai/result
From 2010.igem.org
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Go to [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2010&group=SJTU-BioX-Shanghai Partsregistry] to see our submitted parts. | Go to [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2010&group=SJTU-BioX-Shanghai Partsregistry] to see our submitted parts. | ||
==Demonstration== | ==Demonstration== | ||
| - | + | ===Eukaryotic approach=== | |
| + | #Demonstration of Channelrhodopsin-2 (ChR2) | ||
| + | ##Expression | ||
| + | We transfected ECHO cells with pcDNA containing the gene encoding ChR2 and YFP, and tested the expression of ChR2 by exposing the transfected cells to blue light. Cells were cultured in '''(blank)''', with foil wrapped around to avoid light. 24 hours after transfection, the cells were dyed with '''(blank)''', which means the nuclei would display the color of blue under microscope. With a confocal microscope, we found that blue points(i.e. cell nuclei) were surrounded by green areas, as shown below: | ||
| + | |||
| + | <html><img style="margin:3%;" src="https://static.igem.org/mediawiki/2010/1/12/SJTU10-res-ChR2.png" alt="ChR2 expression 1" title="ChR2 expression 1" /> | ||
| + | </html><html><img style="margin:3%;" src="https://static.igem.org/mediawiki/2010/2/2f/SJTU10-res-ChR2-2.png" alt="ChR2 expression 2" title="ChR2 expression 2" /> | ||
| + | </html> | ||
| + | |||
| + | According to the two pictures above, we saw that ChR2 had been expressed in 293T cell, and even their position was on the cell membrane. This is an essential precondition for its functioning. | ||
| + | |||
| + | ##Function | ||
| + | We then began to test whether this molecule could function well as in neurons. Similar to that in neurons, the expression level of two genes ARC and ZIF268, is the standard for evaluating functioning of ChR2. According to references, we found that intermittent light was better for ChR2 to function normally, whereas continuous light could inactive ChR2. The equipment for discontinuous blue light is like that: | ||
| + | |||
| + | '''(blank)''' | ||
| + | |||
| + | Once started, it illuminated the cell culture in a way similar to that below: | ||
| + | |||
| + | '''(blank)''' | ||
| + | |||
| + | After excitation, we extracted total RNA with a Promega Kit. Then we conducted RT-PCR for ARC and ZIF268 with GAPDH as the reference gene. Photos for the results: | ||
| + | |||
| + | <html><a href="https://static.igem.org/mediawiki/2010/b/b0/SJTU10-res-ChR2-RT-PCR.png"><img width="100%" src="https://static.igem.org/mediawiki/2010/b/b0/SJTU10-res-ChR2-RT-PCR.png" alt="RNA extraction and RT-PCR" title="RNA extraction and RT-PCR" /></a></html> | ||
| + | |||
| + | From the PCR results we found that the expression of ARC had NO evident differences, while that of ZIF268 did. In Fig. 4, cells with blue light illumination has a higher expression level of ZIF268, which indicated the functioning of ChR2. | ||
| + | |||
| + | ##Real-time PCR | ||
| + | |||
| + | In order to render the result more convincing, we also conducted Real-Time PCR for these two genes. | ||
| + | |||
| + | '''(blank)''' | ||
| + | |||
| + | #Demonstration for MEF2-JeT promoter | ||
| + | |||
| + | '''(blank)''' | ||
| + | |||
| + | #Demonstration for ChR2-MEF2-JeT | ||
| + | |||
| + | '''(blank)''' | ||
| + | |||
==Characterization== | ==Characterization== | ||
* '''details''' | * '''details''' | ||
{{Template:SJTU10-footer}} | {{Template:SJTU10-footer}} | ||
Revision as of 07:23, 25 October 2010
Contents |
Result overview
- details
Parts
Go to [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2010&group=SJTU-BioX-Shanghai Partsregistry] to see our submitted parts.
Demonstration
Eukaryotic approach
- Demonstration of Channelrhodopsin-2 (ChR2)
- Expression
We transfected ECHO cells with pcDNA containing the gene encoding ChR2 and YFP, and tested the expression of ChR2 by exposing the transfected cells to blue light. Cells were cultured in (blank), with foil wrapped around to avoid light. 24 hours after transfection, the cells were dyed with (blank), which means the nuclei would display the color of blue under microscope. With a confocal microscope, we found that blue points(i.e. cell nuclei) were surrounded by green areas, as shown below:
According to the two pictures above, we saw that ChR2 had been expressed in 293T cell, and even their position was on the cell membrane. This is an essential precondition for its functioning.
- Function
We then began to test whether this molecule could function well as in neurons. Similar to that in neurons, the expression level of two genes ARC and ZIF268, is the standard for evaluating functioning of ChR2. According to references, we found that intermittent light was better for ChR2 to function normally, whereas continuous light could inactive ChR2. The equipment for discontinuous blue light is like that:
(blank)
Once started, it illuminated the cell culture in a way similar to that below:
(blank)
After excitation, we extracted total RNA with a Promega Kit. Then we conducted RT-PCR for ARC and ZIF268 with GAPDH as the reference gene. Photos for the results:
From the PCR results we found that the expression of ARC had NO evident differences, while that of ZIF268 did. In Fig. 4, cells with blue light illumination has a higher expression level of ZIF268, which indicated the functioning of ChR2.
- Real-time PCR
In order to render the result more convincing, we also conducted Real-Time PCR for these two genes.
(blank)
- Demonstration for MEF2-JeT promoter
(blank)
- Demonstration for ChR2-MEF2-JeT
(blank)
Characterization
- details
