Difference between revisions of "Part:BBa K1720002"
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[[File:HRE-PCR.png|400px|thumb|left|Fig.4  ΔCт Vs. GAPDH of hypoxia responsive promotor under different situation]]
 
[[File:HRE-PCR.png|400px|thumb|left|Fig.4  ΔCт Vs. GAPDH of hypoxia responsive promotor under different situation]]
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Revision as of 01:03, 19 September 2015

Hypoxia-induced promotor

This part is a hypoxia response promotor.We used part BBa_K747096 as a backbone and inserted a hypoxia responsive element to the CMV promotor, so that the CMV promotor will work only under hypoxia situation. 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 .The cells were either cultured under normoxia situation or treated with sodium hyposulfite, an oxygen cleaner to cause hypoxia situation, for 2 hours. As a control, HEK293 cells were also transiently transfected with carrying the original CMV promoter, submitted by the team Freiburg in 2012, followed by the EGFP reporter.

Usage and Biology

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 only observed 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.


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

















Unfortunately, we observed EGFP expression under the control of hypoxia responsive promotor even under aerobic situation. Thus, this device was not a strict hypoxia responsive promotor as we expected.


Fig.4 ΔCт Vs. GAPDH of hypoxia responsive promotor under different situation






























Fig.5 ΔCт Vs. GAPDH of hypoxia responsive promotor and CMV promotor under normoxia situation





We used real time PCR to compare GFP expression level of different groups .The results indicated that under hypoxia situation hypoxia responsive CMV promotor works better than CMV promotor, while under normoxia situation CMV promotor works better.So we planned improve this promotor and make it more sensitive in our future work.We planned improve this promotor in our future work.