Difference between revisions of "Part:BBa K1446003:Experience"
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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. | 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. | 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, we picked a threshold for the green channel at 305 and measured | + | For the image analysis, we used the ImageJ software: After splitting the channels, we picked a threshold for the green channel at 305 (arbitrary fluorescence units) and measured the flurescence for particle sizes between 300 and 1900 pixel and a circularity above 0.01. This selection allowed us to exclude cells that are about to divide or generally not normal in shape or size. |
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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> | 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> | ||
Revision as of 10:59, 17 October 2014
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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, we picked a threshold for the green channel at 305 (arbitrary fluorescence units) and measured the flurescence for particle sizes between 300 and 1900 pixel and a circularity above 0.01. This selection allowed us to exclude cells that are 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.
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