Team:SJTU-BioX-Shanghai/result
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====PfdhF, the hypoxia-inducible promoter==== | ====PfdhF, the hypoxia-inducible promoter==== | ||
- | We tested the hypoxia-inducible promoter, fdhF promoter (PfdfF, | + | We tested the hypoxia-inducible promoter, fdhF promoter (PfdfF, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K387003 Part:BBa_K387003]). In this test experiment, we expected to see that, at the same OD<sub>600</sub>, the activity of PfdhF is higher in hypoxia than in normal oxygen pressure (20%). At the beginning, both the hypoxia group and control (20% oxygen partial pressure, i.e. in air) group were incubated with 200 microlitre suspension of bacteria with the testing part (PfdhF followed by firefly luciferase). Then, we sampled both group and measured OD<sub>600</sub> and value of luciferase every 30 minutes (control group) or 1 hour (hypoxia). The figure was the result of this test. Our result showed that this part worked as we expected: under hypoxia, the efficiency of this promoter was much higher than that under the condition of 20% oxygen at the same OD<sub>600</sub>. (One thing that needs to be noticed is that at 0.4 OD<sub>600</sub>, the bacteria is in log phase and the PfdhF begins to work.) |
<html><a href="https://static.igem.org/mediawiki/2010/8/8b/SJTU10-res-Pfdhf.gif"><img src="https://static.igem.org/mediawiki/2010/8/8b/SJTU10-res-Pfdhf.gif" alt="Hypoxia vs Normal" title="Hypoxia vs Normal" style="width:50%;margin-left:25%;" /></a></html> | <html><a href="https://static.igem.org/mediawiki/2010/8/8b/SJTU10-res-Pfdhf.gif"><img src="https://static.igem.org/mediawiki/2010/8/8b/SJTU10-res-Pfdhf.gif" alt="Hypoxia vs Normal" title="Hypoxia vs Normal" style="width:50%;margin-left:25%;" /></a></html> |
Revision as of 14:29, 26 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)
Prokaryotic approach
PfdhF, the hypoxia-inducible promoter
We tested the hypoxia-inducible promoter, fdhF promoter (PfdfF, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K387003 Part:BBa_K387003]). In this test experiment, we expected to see that, at the same OD600, the activity of PfdhF is higher in hypoxia than in normal oxygen pressure (20%). At the beginning, both the hypoxia group and control (20% oxygen partial pressure, i.e. in air) group were incubated with 200 microlitre suspension of bacteria with the testing part (PfdhF followed by firefly luciferase). Then, we sampled both group and measured OD600 and value of luciferase every 30 minutes (control group) or 1 hour (hypoxia). The figure was the result of this test. Our result showed that this part worked as we expected: under hypoxia, the efficiency of this promoter was much higher than that under the condition of 20% oxygen at the same OD600. (One thing that needs to be noticed is that at 0.4 OD600, the bacteria is in log phase and the PfdhF begins to work.)
Then we wanted to know the difference of PfdhF activity in these two conditions. So, we plotted another figure with OD600 as x axis and the ratio of PfdhF activity under hypoxia to that under normal oxygen pressure as y axis. This curve showed that, during and after the log phase (OD600>0.4), the activity of PfdhF is over 1.5 times more under hypoxia than under 20% oxygen. Besides, this ratio is over 3 when OD600>0.5. Thus, we proved that PfdhF is a hypoxia-inducible promoter with much higher activity under hypoxia than under 20% oxygen.
Characterization
- details