Difference between revisions of "Part:BBa K1720002"
 
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<partinfo>BBa_K1720002 short</partinfo>
 
<partinfo>BBa_K1720002 short</partinfo>
  
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This is a part used for coding soluble human guanylate cyclase subunit:Alpha 3.Alpha 3 unit interacts with a beta subunit to form the guanylate cyclase enzyme.
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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.
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Soluble guanylate cyclases(sGC) are heterodimeric proteins that catalyze the conversion of GTP to 3',5'-cyclic GMP and pyrophosphate. It is important for smooth muscle relaxation in the cardiovascular system. sGC contains an HNOX domain, which serves as a receptor for ligands such as nitric oxide, oxygen and nitrovasodilator drugs.There are several different isoforms of sGC subunit ,but the cardiovascular system abundant with alpha 3 unit and beta 3 unit.  
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Before using this part please add a prompter, in our project we add an EF1A promoter to this part.Since EF1A contains Pstl site,we did not add it to this part.
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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.
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==Experimental Results==
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This part with a GFP reporter was then transfected into HEK293 cells by lentiviral Vector and in vivo green fluorescence signal was observed under fluorescence microscope, it meant that this part was transfected into HEK293 cells successfully.Then we used qRT-PCR to observe transcriptional level of alpha 3 unit.Finally we detected the cGMP concentration with Elisa Kit. And the results are as follow:
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===Improved Results===
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*<b>Group:</b> LZU-CHINA 2019
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<!-- Add more about the biology of this part here
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*<b>Author</b>: Jian Qi; Haodong Yan
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*<b>Summary</b>: We modified a new HRE opereon with 5 reapts following miniCMV promoter(BBa_K2796052). We also had detected the function by monitoring the relative fluorescence intensity of copGFP with different CoCl(II) concentration.We use this part to do our experiment.
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[[File:T--LZU-CHINA--Hif(0).png|600px|thumb|center|]]
 
===Usage and Biology===
 
===Usage and Biology===
  
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<partinfo>BBa_K1720002 parameters</partinfo>
 
<partinfo>BBa_K1720002 parameters</partinfo>
 
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Before we start the experiment, we add our part to psb1c3 vector and packaged the vector with lipo2000.
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===Vector Map：===
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[[File:SCUT2015 China HRE Vector.png|600px|thumb|left|Fig.1]]
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===Experiment：===
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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.
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<b>Protocol：</b>
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1. Seed cells to be 40% confluent at a 35mm culture dish.
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2. Dilute 2.5ul Lipofectamine2000 Reagent in 50ul Opti-MEM Medium
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3. Dilute 2.5ul (400ng/ul) plasmids in 50ul Opti-MEM Medium
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4. Mix diluted Lipofectamine2000 Reagent with diluted plasmids, incubating for 5 min.
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5. Withdraw culture medium from 35mm culture dish.
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6. Add 1ml Opti-MEM Medium and plasmid-Lipo complex to cells
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7. Incubate for 15 hours.
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8. Withdraw medium from culture dish.
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9. Add 2ml DMEM medium( containing 10% FBS )to cells and incubate for 10 hours
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10. Withdraw culture medium from 35mm culture dish.
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11. Add 20ul sodium hyposulfite(100umol/L ) to cells
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12. Incubate for 1 hour.
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13. Observe the cells under Inverted fluorescence microscope.
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<b>Result:</b>
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[[File:SCUT2015 China CMV transfection.png|400px|thumb|left|Fig.2  EGFP signal under the control of orginal CMV promotor in normoxia situation]]
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[[File:SCUT2015 China HRE transfection2.png|400px|thumb|left|Fig.3 EGFP signal under the regulate of hypoxia responsive CMV in normoxia situation ]]
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[[File:SCUT2015 China HRE-1 transfection.png|400px|thumb|left|Fig.4  EGFP signal under the regulate of  hypoxia responsive CMV in hypoxia situation]]
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Under normoxia condition, we observed weaker green fluorescence under the control of HRE, the hypoxia responsive promotor, than that under the control of the original CMV promoter. Moreover, under the control of HRE, more cells exhibited green fluorescence under hypoxia condition than under normoxia condition. The results suggested that our HRE is working.
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[[File:HRE-PCR.png|400px|thumb|left|Fig.4  ΔCт Vs. GAPDH of hypoxia responsive promotor under different situation]]
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[[File:HRE-PCR2.png|400px|thumb|left|Fig.5 ΔCт Vs. GAPDH of hypoxia responsive promotor and CMV promotor under normoxia situation]]
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The overall florescent intensity under the control of HRE, however, were similar between hypoxia and normoxia conditions. After discussion, we thought there may be several issues in the model, i.e. slower growth of cells or weaker activity of EGFP under the hypoxia condition. Thus, we also measured expression of EGFP by real-time PCR. That data further proved that the HRE device worked as we expected, although not as strict. We believe we can improve this promotor and make it more sensitive in our future work.
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===References===
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<partinfo>BBa_K2295003</partinfo> was sent in 2017 by Team Freiburg as an improvement of this BioBrick.

Latest revision as of 00:22, 22 October 2019

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.

Experimental Results

Improved Results

  • Group: LZU-CHINA 2019
  • Author: Jian Qi; Haodong Yan
  • Summary: We modified a new HRE opereon with 5 reapts following miniCMV promoter(BBa_K2796052). We also had detected the function by monitoring the relative fluorescence intensity of copGFP with different CoCl(II) concentration.We use this part to do our experiment.
T--LZU-CHINA--Hif(0).png

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 orginal CMV promotor in normoxia situation
















Fig.3 EGFP signal under the regulate of hypoxia responsive CMV in normoxia situation
















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

















Under normoxia condition, we observed weaker green fluorescence under the control of HRE, the hypoxia responsive promotor, than that under the control of the original CMV promoter. Moreover, under the control of HRE, more cells exhibited green fluorescence under hypoxia condition than under normoxia condition. The results suggested that our HRE is working.



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






The overall florescent intensity under the control of HRE, however, were similar between hypoxia and normoxia conditions. After discussion, we thought there may be several issues in the model, i.e. slower growth of cells or weaker activity of EGFP under the hypoxia condition. Thus, we also measured expression of EGFP by real-time PCR. That data further proved that the HRE device worked as we expected, although not as strict. We believe we can improve this promotor and make it more sensitive in our future work.











References

BBa_K2295003 was sent in 2017 by Team Freiburg as an improvement of this BioBrick.