Difference between revisions of "Part:BBa K092600:Experience"
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | ||
how you used this part and how it worked out. | how you used this part and how it worked out. | ||
| − | === | + | ===Characterization of BBa_K092600=== |
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| + | Theoretically this construct should not be able to express RFP due to the fact that the promoter for the induction molecule (TetR) is not present and thus no TetR is expressed. | ||
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| + | In the contrary, we found the expression of TetR to be leaky and we carried out characterization of the part. (see protocol in parts registry BBa_K092600). | ||
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| + | We found that RPF was indeed produced thus indicating a basal ‘leaky’ expression, and this allows us to predict that upon addition of the PLac promotor, the production of RFP will be highly enhanced. | ||
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| + | We obtained a transfer function that is shown below. | ||
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| + | [[Image:Transfer_function.jpg]] | ||
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| + | Flow cytometry was carried out to verify the presence of RFP expressing cells. After the plate reading assay, the same samples were taken for flow cytometry. Below the flow cytometry for samples at two high concentrations of is shown. In the histograms below, we can clearly see the shift in the different cell types, between those that produce and do not produce RFP. In the x-axis is the log of fluorescence intensity, and in the y-axis the cell count. | ||
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| + | [[Image:Cytometer.jpg]] | ||
| + | These results demonstrate that the construct that was made does indeed function and that upon completion can give high levels of RFP. At the same time such production of RFP can interfere with the precise control that is required in our system. | ||
===User Reviews=== | ===User Reviews=== | ||
Revision as of 23:46, 29 October 2008
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Characterization of BBa_K092600
Theoretically this construct should not be able to express RFP due to the fact that the promoter for the induction molecule (TetR) is not present and thus no TetR is expressed.
In the contrary, we found the expression of TetR to be leaky and we carried out characterization of the part. (see protocol in parts registry BBa_K092600).
We found that RPF was indeed produced thus indicating a basal ‘leaky’ expression, and this allows us to predict that upon addition of the PLac promotor, the production of RFP will be highly enhanced.
We obtained a transfer function that is shown below.
Flow cytometry was carried out to verify the presence of RFP expressing cells. After the plate reading assay, the same samples were taken for flow cytometry. Below the flow cytometry for samples at two high concentrations of is shown. In the histograms below, we can clearly see the shift in the different cell types, between those that produce and do not produce RFP. In the x-axis is the log of fluorescence intensity, and in the y-axis the cell count.
These results demonstrate that the construct that was made does indeed function and that upon completion can give high levels of RFP. At the same time such production of RFP can interfere with the precise control that is required in our system.
User Reviews
UNIQfef033587248ee5c-partinfo-00000000-QINU UNIQfef033587248ee5c-partinfo-00000001-QINU