Difference between revisions of "Part:BBa K415511"

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	__NOTOC__		__NOTOC__
	<partinfo>BBa_K415511 short</partinfo>		<partinfo>BBa_K415511 short</partinfo>
−	MIT iGEM 2010 Mammalian Part	+	L4R1 MammoBlock Promoter Vector. The PAI2 (Plasminogen Activator Inhibitor 2) promoter is suspected of containing regulatory mechanosensory elements, specifically cyclic, nonuniform strain. The sequence from the human PAI2 [-2000,249], where 0 designates the transcriptional start site, was isolated via genomic PCR.
		+
		+	==Characterization==
		+
		+	[[Image:PAI2_characterization.png|thumb|left|Figure 1. Shear stress response of PAI2_EGFP.]] Response of K415511 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 pPAI2_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, PAI2 experienced significant upregulation in transcriptional activity early in the experiment at 8hr (2.5x) under low level shear stress, as compared to negative control, which experienced no shear stress. However, overall expression of EGFP was very low throughout the experiment.
		+
		+	PAI2 was not expected to respond to fluid shear stress but primarily to cyclic, nonuniform mechanical strain. We believe that while the early activation of this promoter may be significant, more experiments in cyclic nonuniform strain will produce more striking results.
−	<!-- Add more about the biology of this part here
−	===Usage and Biology===
−	<!-- -->
	<span class='h3bb'>Sequence and Features</span>		<span class='h3bb'>Sequence and Features</span>
	<partinfo>BBa_K415511 SequenceAndFeatures</partinfo>		<partinfo>BBa_K415511 SequenceAndFeatures</partinfo>

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Latest revision as of 22:21, 28 October 2010

pPAI2 L4R1 MammoBlock Entry Vector

L4R1 MammoBlock Promoter Vector. The PAI2 (Plasminogen Activator Inhibitor 2) promoter is suspected of containing regulatory mechanosensory elements, specifically cyclic, nonuniform strain. The sequence from the human PAI2 [-2000,249], where 0 designates the transcriptional start site, was isolated via genomic PCR.

Characterization

Response of K415511 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 pPAI2_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, PAI2 experienced significant upregulation in transcriptional activity early in the experiment at 8hr (2.5x) under low level shear stress, as compared to negative control, which experienced no shear stress. However, overall expression of EGFP was very low throughout the experiment.

PAI2 was not expected to respond to fluid shear stress but primarily to cyclic, nonuniform mechanical strain. We believe that while the early activation of this promoter may be significant, more experiments in cyclic nonuniform strain will produce more striking results.

Sequence and Features