Difference between revisions of "Part:BBa K1720002"

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<b>Result:</b>
 
<b>Result:</b>
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[[File:SCUT2015 China CMV transfection.png|400px|thumb|left|Fig.2  Positive Control Group]]
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[[File:SCUT2015 China CMV transfection.png|400px|thumb|left|Fig.2  EGFP signal under the control of CMV promotor]]
  
  
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[[File:SCUT2015 China HRE transfection2.png|400px|thumb|left|Fig.3   Control Group]]
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[[File:SCUT2015 China HRE transfection2.png|400px|thumb|left|Fig.3 EGFP signal under the regulate of HRE in aerobic situation ]]
  
  
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[[File:SCUT2015 China HRE-1 transfection.png|400px|thumb|left|Fig.4  Experimental Group]]
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[[File:SCUT2015 China HRE-1 transfection.png|400px|thumb|left|Fig.4  EGFP signal under the regulate of HRE in hypoxia situation]]
  
  
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From the results above, we can see that hypoxia responsive promotor still working under aerobic situation. So that this promotor is not a strict hypoxia responsive promotor as we expect. And we used RT-PCR to see whether this promotor works better under hypoxia situation.
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From the results above, we can see that hypoxia responsive promotor still working under aerobic situation. So that this promotor is not a strict hypoxia responsive promotor as we expect.So we will imoprove this part in the future and make it more sensitive.

Revision as of 02:30, 18 September 2015

Hypoxia-induced promotor

This part is a hypoxia response element.When the cells suffer from hypoxia situation this element will begin to work.It will activate the downstream gene expression.The hypoxia response element is a minimal cis-regulatory element mediating transactivation by the hypoxia-inducible factor (HIF) in mammalian cells.

This element with a GFP reporter was then transfected into HEK293 cells .Then we treat the cells with sodium hyposulfite, an reagent that leads to hypoxia. The negative control was HEK293 cells that treat with PBS and we did not detect green fluorescence signal.But we detected green fluorescence signal in the experimental group ,which meant that this element work under hypoxia situation.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 41
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 41
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 41
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 41
  • 1000
    COMPATIBLE WITH RFC[1000]



Before we start the experiment, we add our part to psb1c3 vector and packaged the vector with lipo2000.


Vector Map：

Fig.1























Experiment：

We transiently transfected HEK293 cells with plasmids containing hypoxia-induced promotor and EGFP reporter. The cells were treated with sodium hyposulfite, an oxygen cleaner to cause hypoxia situation.If green fluorescence signal was observed only in experimental group and positive control, our Hypoxia-induced promotor’s function will be demonstrated.


Protocol：

1. Seed cells to be 40% confluent at a 35mm culture dish.

2. Dilute 2.5ul Lipofectamine2000 Reagent in 50ul Opti-MEM Medium

3. Dilute 2.5ul (400ng/ul) plasmids in 50ul Opti-MEM Medium

4. Mix diluted Lipofectamine2000 Reagent with diluted plasmids, incubating for 5 min.

5. Withdraw culture medium from 35mm culture dish.

6. Add 1ml Opti-MEM Medium and plasmid-Lipo complex to cells

7. Incubate for 15 hours.

8. Withdraw medium from culture dish.

9. Add 2ml DMEM medium( containing 10% FBS )to cells and incubate for 10 hours

10. Withdraw culture medium from 35mm culture dish.

11. Add 20ul sodium hyposulfite(100umol/L ) to cells

12. Incubate for 1 hour.

13. Observe the cells under Inverted fluorescence microscope.


Note: The negative control was transiently transfected with the same plasmids in experimental group but did not treat with sodium hyposulfite. The positive control was transiently transfected with plasmids that contain CMV promoter and EGFP reporter.

Result:

Fig.2 EGFP signal under the control of CMV promotor















Fig.3 EGFP signal under the regulate of HRE in aerobic situation















Fig.4 EGFP signal under the regulate of HRE in hypoxia situation

















From the results above, we can see that hypoxia responsive promotor still working under aerobic situation. So that this promotor is not a strict hypoxia responsive promotor as we expect.So we will imoprove this part in the future and make it more sensitive.