Difference between revisions of "Part:BBa K1446003:Experience"
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===Applications of BBa_K1446003=== | ===Applications of BBa_K1446003=== | ||
| − | We cloned this part with an mCherry fusion at the C terminus of the dcmR gene | + | We cloned this part with an mCherry fusion at the C terminus of the dcmR gene in the pOXON-1 plamsid (pME6010 plasmid with kanamycin resistance). Expression was induced with various amounts of ATC and the following fluorescence data acquired. Exposure time was 0.2 seconds. As no calibration data was obtained using purified mCherry, the results have been left in fluorescence arbitrary units. |
| − | + | The liquid cultures were grown overnight (16 hours). In the morning, 1 in 200 dilutions were made (with fresh antibiotics) and concentrations of 0, 10, 50, 100 and 200 ng/uL ATC. | |
| − | Expression was induced with various amounts of ATC and the following fluorescence data acquired. Exposure time was 0.2 seconds. As no calibration data was obtained using purified mCherry, the results have been left in fluorescence arbitrary units.<br><br> | + | For the image analysis, we used the ImageJ software: After splitting the channels (into red, green and blue), we picked a threshold for the green channel at 305 (arbitrary fluorescence units) and measured the fluorescence for particle sizes between 300 and 1900 pixel and a circularity above 0.01. This selection allowed us to exclude cells that are aggregated together, about to divide or generally not normal in shape or size. |
| + | <br> | ||
| + | mCherry fluorescence increases with amount of ATC used confirming that the dcmR gene was expressed under the control of the tet promoter and operator system.<br><br> | ||
[[File:Oxford_DcmR-mCherry_expression_induced_by_0ng_ATC.png|centre|750px]] | [[File:Oxford_DcmR-mCherry_expression_induced_by_0ng_ATC.png|centre|750px]] | ||
[[File:Oxford_DcmR-mCherry_expression_induced_by_10ng_ATC.png|centre|750px]] | [[File:Oxford_DcmR-mCherry_expression_induced_by_10ng_ATC.png|centre|750px]] | ||
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[[File:Oxford_DcmR-mCherry_expression_induced_by_200ng_ATC.png|centre|750px]] | [[File:Oxford_DcmR-mCherry_expression_induced_by_200ng_ATC.png|centre|750px]] | ||
[[File:Oxford_Comparison_of_mean_flouresence_intensity_from_DcmR-mCherry_expression_induced_at_varying_amounts_of_ATC.png|centre|900px]] | [[File:Oxford_Comparison_of_mean_flouresence_intensity_from_DcmR-mCherry_expression_induced_at_varying_amounts_of_ATC.png|centre|900px]] | ||
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===User Reviews=== | ===User Reviews=== | ||
<!-- DON'T DELETE --><partinfo>BBa_K1446003 StartReviews</partinfo> | <!-- DON'T DELETE --><partinfo>BBa_K1446003 StartReviews</partinfo> | ||
Latest revision as of 11:02, 17 October 2014
This experience page is provided so that any user may enter their experience using this part.
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how you used this part and how it worked out.
Applications of BBa_K1446003
We cloned this part with an mCherry fusion at the C terminus of the dcmR gene in the pOXON-1 plamsid (pME6010 plasmid with kanamycin resistance). Expression was induced with various amounts of ATC and the following fluorescence data acquired. Exposure time was 0.2 seconds. As no calibration data was obtained using purified mCherry, the results have been left in fluorescence arbitrary units.
The liquid cultures were grown overnight (16 hours). In the morning, 1 in 200 dilutions were made (with fresh antibiotics) and concentrations of 0, 10, 50, 100 and 200 ng/uL ATC.
For the image analysis, we used the ImageJ software: After splitting the channels (into red, green and blue), we picked a threshold for the green channel at 305 (arbitrary fluorescence units) and measured the fluorescence for particle sizes between 300 and 1900 pixel and a circularity above 0.01. This selection allowed us to exclude cells that are aggregated together, about to divide or generally not normal in shape or size.
mCherry fluorescence increases with amount of ATC used confirming that the dcmR gene was expressed under the control of the tet promoter and operator system.
User Reviews
UNIQb3b8480804d6b1c0-partinfo-00000000-QINU UNIQb3b8480804d6b1c0-partinfo-00000001-QINU