Difference between revisions of "Part:BBa J04450: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. | ||
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| + | Our aim was to evaluate RFP expression quantity of E. coli. In order to obtain this information, we agreed that the measurement of fluorescence intensity in certain OD values is the best approach. However, fluorescence quenching was a setback which we must overcome for accurate and reliable results. Because, as a result of quenching, fluorescence intensity counts do not give the actual amount of fluorescence expected from a certain OD value. Therefore we employed a curve fitting method suggested by Zhang et. al (2010) which is claimed to take the quenching problem into consideration | ||
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| + | <html> <div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 162px; height: 34px;" alt="w7" src="https://static.igem.org/mediawiki/2010/2/2b/Denklem_1.jpg"></a></div> </html> | ||
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| + | <br> Where If is the fluorescence intensity; OD is the cell density; y0 is the offset; A1 is the amplitude; and t1 is the decay constant. | ||
| + | <br> We chose BBa_J04450 for this experiment. And our negative control was again BBa_B0034. | ||
| + | <br> There were nine bacterial concentrations prepared for one to five hours of IPTG induction. (All concentrations were prepared as triplicates.) Then a complete graph of all the induction durations was plotted with the help of the function shown above: | ||
| + | <br> | ||
| + | <html><div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 280px;" alt="w7" src="https://static.igem.org/mediawiki/2010/9/9b/C7.jpg"></a></div> </html> | ||
| + | <br> <div align="center">(■) 1 h induced; (●) 2 h induced; (▲) 3 h induced; (▼) 4 h induced; (♦) 5 h induced </div> | ||
| + | <br> | ||
| + | <br> As seen in the graph, fluorescence behaves exactly as expected and does not increase linearly with the increased bacterial concentration. However, IPTG induction effect is clear on fluorescence intensity. (Error bars were too small to be visible.) | ||
| + | <br> | ||
| + | <html> <div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 314px; height: 210px;" alt="w7" src="https://static.igem.org/mediawiki/2010/b/b0/Fluorescence_intensity.jpg"></a></div> </html> | ||
| + | <br> | ||
| + | <br> Finally, the variables A1 and t1 of the fittings of each induction duration were used to obtain a mean fluorescence intensity (MFI) value: | ||
| + | <html> <div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 136px; height: 42px;" alt="w7" src="https://static.igem.org/mediawiki/2010/d/d6/MFI.jpg"></a></div> </html> | ||
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| + | <html> <div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 300px; height: 280px;" alt="w7" src="https://static.igem.org/mediawiki/2010/c/c2/C8.jpg"></a></div> </html> | ||
| + | <br> | ||
| + | <html> <div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 266px; height: 135px;" alt="w7" src="https://static.igem.org/mediawiki/2010/3/32/MFI_table.jpg"></a></div> </html> | ||
| + | <br> | ||
| + | <br>By conducting this experiment, we were able to characterize RFP expression pattern of the part BBa_J04450 in the host Top Ten. Theoretical mean fluorescence intensity reflects the fluorescence quantum efficiency of the fluorophore RFP, and was found to be unrelated to the cell concentration. Since it is obvious from the results we have obtained that change in the OD does not correlate linearly with the change in the fluorescence intensity. Therefore this model proposes a solution to the unpredictability of the fluorescence amount coming from a certain bacterial population. | ||
| + | |||
| + | The characterization was performed by METU-TURKEY 2010 IGEM TEAM. | ||
===Applications of BBa_J04450=== | ===Applications of BBa_J04450=== | ||
Revision as of 00:23, 28 October 2010
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.
Our aim was to evaluate RFP expression quantity of E. coli. In order to obtain this information, we agreed that the measurement of fluorescence intensity in certain OD values is the best approach. However, fluorescence quenching was a setback which we must overcome for accurate and reliable results. Because, as a result of quenching, fluorescence intensity counts do not give the actual amount of fluorescence expected from a certain OD value. Therefore we employed a curve fitting method suggested by Zhang et. al (2010) which is claimed to take the quenching problem into consideration
Where If is the fluorescence intensity; OD is the cell density; y0 is the offset; A1 is the amplitude; and t1 is the decay constant.
We chose BBa_J04450 for this experiment. And our negative control was again BBa_B0034.
There were nine bacterial concentrations prepared for one to five hours of IPTG induction. (All concentrations were prepared as triplicates.) Then a complete graph of all the induction durations was plotted with the help of the function shown above:
As seen in the graph, fluorescence behaves exactly as expected and does not increase linearly with the increased bacterial concentration. However, IPTG induction effect is clear on fluorescence intensity. (Error bars were too small to be visible.)
Finally, the variables A1 and t1 of the fittings of each induction duration were used to obtain a mean fluorescence intensity (MFI) value:
By conducting this experiment, we were able to characterize RFP expression pattern of the part BBa_J04450 in the host Top Ten. Theoretical mean fluorescence intensity reflects the fluorescence quantum efficiency of the fluorophore RFP, and was found to be unrelated to the cell concentration. Since it is obvious from the results we have obtained that change in the OD does not correlate linearly with the change in the fluorescence intensity. Therefore this model proposes a solution to the unpredictability of the fluorescence amount coming from a certain bacterial population.
The characterization was performed by METU-TURKEY 2010 IGEM TEAM.
Applications of BBa_J04450
User Reviews
UNIQ16c7c42576e17ced-partinfo-00000006-QINU
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Antiquity |
This review comes from the old result system and indicates that this part worked in some test. |
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We've used this part as an alternative to the suicide part p1010 (ccdB) for selecting against cells that have been transformed with a [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf destination plasmid]. It worked perfectly, and if i might say, it looks smashingly in LB medium ;). |
UNIQ16c7c42576e17ced-partinfo-00000009-QINU