Team:SJTU-BioX-Shanghai/result

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(PfdhF, the hypoxia-inducible promoter)
(PfdhF, the hypoxia-inducible promoter)
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<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:70%;margin-left:15%;" /></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:70%;margin-left:15%;" /></a></html>
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Then we wanted to know the difference of PfdhF activity in these two conditions. So, we plotted another figure with OD<sub>600</sub> 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 (OD<sub>600</sub>>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 OD<sub>600</sub>>0.5. Thus, we proved that PfdhF is a hypoxia-inducible promoter with much higher activity under hypoxia than under 20% oxygen.
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Then we wanted to know the difference of PfdhF activity under these two conditions. So, we plotted another figure with OD<sub>600</sub> 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 normal 20% oxygen pressure. Particularly, this ratio is over 3 when OD<sub>600</sub>>0.5. Furthermore, in the normal 20% oxygen group, as the OD<sub>600</sub> increases, the oxygen supplies may be insufficient for <i>E. coli</i> which may lead to the increase of PfdhF activity. In this case, the activity of PfdhF under normal condition is probably overestimated. With all these data and analysis, it is evident that PfdhF is a rather good hypoxia-inducible promoter with much higher activity under hypoxia than under 20% oxygen pressure.
<html><a href="https://static.igem.org/mediawiki/2010/9/9c/SJTU10-res-Pfdhf2.gif"><img src="https://static.igem.org/mediawiki/2010/9/9c/SJTU10-res-Pfdhf2.gif" alt="Ratio of Hypoxia to Normal" title="Ratio of Hypoxia to Normal" style="width:70%;margin-left:15%;" /></a></html>
<html><a href="https://static.igem.org/mediawiki/2010/9/9c/SJTU10-res-Pfdhf2.gif"><img src="https://static.igem.org/mediawiki/2010/9/9c/SJTU10-res-Pfdhf2.gif" alt="Ratio of Hypoxia to Normal" title="Ratio of Hypoxia to Normal" style="width:70%;margin-left:15%;" /></a></html>

Revision as of 11:57, 27 October 2010

SJTU-BioX-Shanghai 2010

Contents

Result overview

  • details

Parts

Go to 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:

ChR2 expression 1 ChR2 expression 2

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:

RNA extraction and RT-PCR

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

Yupeng He built and tested the hypoxia-inducible promoter, fdhF promoter (PfdfF, 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 are in log phase and the PfdhF begins to work.)

Hypoxia vs Normal

Then we wanted to know the difference of PfdhF activity under 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 normal 20% oxygen pressure. Particularly, this ratio is over 3 when OD600>0.5. Furthermore, in the normal 20% oxygen group, as the OD600 increases, the oxygen supplies may be insufficient for E. coli which may lead to the increase of PfdhF activity. In this case, the activity of PfdhF under normal condition is probably overestimated. With all these data and analysis, it is evident that PfdhF is a rather good hypoxia-inducible promoter with much higher activity under hypoxia than under 20% oxygen pressure.

Ratio of Hypoxia to Normal

Characterization

  • details