Difference between revisions of "Part:BBa K415510"
 
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CRE2 is a hybrid promoter, constructed by placing elements from the mechanosensitive FosB promoter in front of an SV40 constitutive promoter. It's been shown that ERK-dependent activation of CREB binding to a CRE/AP-1 like element (designated “CRE2”) in the FosB promoter is one of the main effectors of mechanosensitivity; SRE, or Shear Responsive Element, was first characterized as a mechanosensitive element in the cfos promoter.  
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<partinfo>BBa_K415510 short</partinfo>
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L4R1 MammoBlock Promoter Vector. Preliminary data suggests that this synthetic promoter lends transcriptional sensitivity to fluid shear stress in the HEK293FT cell line. SRE/CRE2_SV40 consists of a concatenation of three elements:
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* SRE, or Shear Responsive Element: was first characterized as a mechanosensitive element in the cfos promoter.
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* CRE2, or CRE/AP-1 like element: mechanosensitive element isolated from the FosB promoter. It has been shown that ERK-dependent activation of CREB binding to a in the FosB promoter is one of the main effectors of mechanosensitivity;  
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* SV40: minimal constitutive promoter.  
  
 
==Characterization==
 
==Characterization==
  
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The response of K415510 to mechanical stimulation via fluid shear stress:
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[[Image:SRE_Characterization.png|thumb|left|Figure 1. Shear stress response of SRE/CRE2_SV40_EGFP.]]
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The response of K415510 to mechanical stress via low level nonuniform fluid shear stress.
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Transient tranfections of pMech_EGFP constructs were performed on HEK293FT cells seeded at 2 million cells/well in two identically seeded six-well plates. The constructs used were pSRE/CRE2_SV40_EGFP and CMV_EGFP. CMV_EGFP served as a control for transformation efficiency.
  
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[[Image:SRE_Characterization.png]]
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12 hours after transfection one of the two six well plates was designated as "shear stress" and placed on a shaker at 120 rpm inside the incubator. The other plate was designated "control" and not subjected to shaking. Fluorescent micrographs were obtained on a Zeiss microscope for each well at 100, 215, and 500 ms exposure times every 3 hours. Brightfield images were exposed for 6 ms.  
  
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Transient tranfections of pMech_EGFP constructs were performed on HEK293FT cells seeded at 2 million cells/well in two identically seeded six-well plates. The constructs used were pSRE/CRE2_SV40_EGFP and CMV_EGFP. CMV_EGFP served as a control for transformation efficiency. 12 hours after transfection one of the two six well plates was designated as "shear stress" and placed on a shaker at 120 rpm inside the incubator. The other plate was designated "control" and not subjected to shaking. Fluorescent micrographs were obtained on a Zeiss microscope for each well at 100, 215, and 500 ms exposure times every 3 hours. Brightfield images were exposed for 6 ms. Images were exported from the accompanying Axiovision software at identical histogram levels for each cell line. For image analysis, accurate cell count could not be obtained because HEK cells grow at high density. Instead area occupied by cells was used as an analogous measurement for fields of view with similar % confluence and measured with imageJ, an image analysis software provided by the NIH. Fluorescent micrographs were processed using the binary function in imageJ, followed by particle analysis to obtain the total area occupied by fluorescence. The ratio of the area of fluorescence and the area of cells were calculated (R_fl) for time points 2.5h, 8h, 15.5h, and 24.5h. The overall ratio R_o was calculated as R_fl(T)/R_fl(2.5), where T=8,15.5, or 24.5. In this process the image levels and cell confluence of each micrograph were carefully matched for comparison.
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Images were exported from the accompanying Axiovision software at identical histogram levels for each cell line. For image analysis, accurate cell count could not be obtained because HEK cells grow at high density. Instead area occupied by cells was used as an analogous measurement for fields of view with similar % confluence and measured with imageJ, an image analysis software provided by the NIH. Fluorescent micrographs were processed using the binary function in imageJ, followed by particle analysis to obtain the total area occupied by fluorescence. The ratio of the area of fluorescence and the area of cells were calculated (R_fl) for time points 2.5h, 8h, 15.5h, and 24.5h. The overall ratio R_o was calculated as R_fl(T)/R_fl(2.5), where T=8,15.5, or 24.5. In this process the image levels and cell confluence of each micrograph were carefully matched for comparison.
  
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'''Results'''
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====Results and Conclusion====
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As seen in Figure 1, SRE/CRE2 exhibited a significant increase in activity (2.4x) compared to the negative control, with no applied shear stress. SRE/CRE2 are elements strongly suspected of being shear stress responsive in literature. Here we provide additional information that our synthetic promoter responds to shear stress by transcriptional upregulation.
  
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SRE/CRE2 exhibited a significant increase in activity (2.4x) compared to the negative control, with no applied shear stress.
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==Sequence and Features==
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<partinfo>BBa_K415510 SequenceAndFeatures</partinfo>

Latest revision as of 02:42, 29 October 2010

pSRE/CRE2_SV40 L4R1 Mammoblock

L4R1 MammoBlock Promoter Vector. Preliminary data suggests that this synthetic promoter lends transcriptional sensitivity to fluid shear stress in the HEK293FT cell line. SRE/CRE2_SV40 consists of a concatenation of three elements:

  • SRE, or Shear Responsive Element: was first characterized as a mechanosensitive element in the cfos promoter.
  • CRE2, or CRE/AP-1 like element: mechanosensitive element isolated from the FosB promoter. It has been shown that ERK-dependent activation of CREB binding to a in the FosB promoter is one of the main effectors of mechanosensitivity;
  • SV40: minimal constitutive promoter.

Characterization

Figure 1. Shear stress response of SRE/CRE2_SV40_EGFP.

The response of K415510 to mechanical stress via low level nonuniform fluid shear stress.

Transient tranfections of pMech_EGFP constructs were performed on HEK293FT cells seeded at 2 million cells/well in two identically seeded six-well plates. The constructs used were pSRE/CRE2_SV40_EGFP and CMV_EGFP. CMV_EGFP served as a control for transformation efficiency.

12 hours after transfection one of the two six well plates was designated as "shear stress" and placed on a shaker at 120 rpm inside the incubator. The other plate was designated "control" and not subjected to shaking. Fluorescent micrographs were obtained on a Zeiss microscope for each well at 100, 215, and 500 ms exposure times every 3 hours. Brightfield images were exposed for 6 ms.

Images were exported from the accompanying Axiovision software at identical histogram levels for each cell line. For image analysis, accurate cell count could not be obtained because HEK cells grow at high density. Instead area occupied by cells was used as an analogous measurement for fields of view with similar % confluence and measured with imageJ, an image analysis software provided by the NIH. Fluorescent micrographs were processed using the binary function in imageJ, followed by particle analysis to obtain the total area occupied by fluorescence. The ratio of the area of fluorescence and the area of cells were calculated (R_fl) for time points 2.5h, 8h, 15.5h, and 24.5h. The overall ratio R_o was calculated as R_fl(T)/R_fl(2.5), where T=8,15.5, or 24.5. In this process the image levels and cell confluence of each micrograph were carefully matched for comparison.

Results and Conclusion

As seen in Figure 1, SRE/CRE2 exhibited a significant increase in activity (2.4x) compared to the negative control, with no applied shear stress. SRE/CRE2 are elements strongly suspected of being shear stress responsive in literature. Here we provide additional information that our synthetic promoter responds to shear stress by transcriptional upregulation.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 6
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 6
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 6
    Illegal BglII site found at 82
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 6
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 6
  • 1000
    COMPATIBLE WITH RFC[1000]