Difference between revisions of "Part:BBa K861060: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. | ||
| + | == Experiment == | ||
| + | <html> | ||
| + | <p>We investigated FadR expression using a Tecan Plate Reader. LB broth containing varying concentrations of oleic acid were innoculated with DH5a <i>E. coli</i> cells expressing fadR (BBa_K861060), fadR-RFP (BBa_K861060), or consituative RFP (BBa_J23110). These were loaded into a clear flat bottomed black walled 96 well plate, some wells contained no cells and were used as blanks. Cells were grown in the Tecan plate reader overnight at 37oC with shaking for a period of 16 hours. OD600 measurements were taken every 30 minutes. In addition RFP readings were also taken with 565nm excitation and 605nm emission wavelengths. </p> | ||
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| + | <p>Values were normalised by OD and are shown as a mean of four repeats.</p> | ||
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| + | <img src="https://static.igem.org/mediawiki/2014/b/bf/Sheffield_2014-0_oleic.png"> | ||
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| + | <img src="https://static.igem.org/mediawiki/2014/7/78/Sheffield_2014_5_oleic.png"> | ||
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| + | <img src="https://static.igem.org/mediawiki/2014/e/ea/Sheffield_2014_10_oleic.png"> | ||
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| + | <img src="https://static.igem.org/mediawiki/2014/f/f4/Sheffield_2014-20_oleic.png"> | ||
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| + | <br> | ||
| + | <p>In the graphs above, a trend can be seen for all concentrations of oleic acid, that fluorescence produced from constitutively expressed RFP increases over time. In comparison there was no observable trend in fadR-RFP expressing cells, showing no increase in fluorescence in any oleic acid concentration. In addition there was no difference in the amount of fluorescence observed between the fadR-RFP and the fadR only and empty cells. </p> | ||
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| + | <p>The FadR promoter and BBa_J23110 constitutive promoter upstream of RFP are the same in strength and so this disregards the observed difference in expression under the two promoters being one based on strength. Further investigation was carried out using fluorescence microscopy to measure RFP fluorescence in response to different oleic acid concentrations and a similar result was observed. </p> | ||
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| + | <p>Based on this information and the results shown above, we suggest that the FadR promoter does not result in expression of RFP downstream in the presence of oleic acid.</p> | ||
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| + | <img style="width:100%" src="https://static.igem.org/mediawiki/2014/c/c3/Picture1.jpg"> | ||
| + | |||
| + | </body> | ||
| + | </html>--[[User:E.court|E.court]] 22:51, 17 October 2014 (CDT) | ||
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===Applications of BBa_K861060=== | ===Applications of BBa_K861060=== | ||
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<h4>Result</h4> | <h4>Result</h4> | ||
<p align="justify"> Normalized using Fluorescence/0D600</p> | <p align="justify"> Normalized using Fluorescence/0D600</p> | ||
| − | <p align="justify"> | + | <p align="justify"> Green: PfadR</p> |
| − | + | <p align="justify"> Glucose Concentration gradient: 1, 10 mM</p> | |
| − | <p align="justify"> Glucose Concentration gradient: | + | <p align="justify"> Fatty acid Concentration gradient: 0.025, 0.05, 0.1, 0.5,1.5 mM</p> |
| − | <p align="justify"> Fatty acid Concentration gradient: 0.025, 0.05, 0.1 | + | |
| − | <p><img src="https://static.igem.org/mediawiki/parts/ | + | <p><img src="https://static.igem.org/mediawiki/parts/6/6f/Pfadr.png" width="500" height="350" hspace="2" vspace="1" border="2" align="top" /></p> |
| − | <p><i> PfadR | + | <p><i> PfadR promoter strength at different glucose and fatty acids concentration</i></p> |
| − | <p align="justify"> As can be shown from the results, the promoter shows about 3 times induction from glucose to 1.5umol/L fatty acids medium and the fluorescence of PfadR | + | <p align="justify"> As can be shown from the results, the promoter shows about 3 times induction from glucose to 1.5umol/L fatty acids medium and the fluorescence of PfadR. Also, in medium that use glucose as sole carbon source, PfadR seems to be leaky. However, since our bacteria is wild type <i>E.coli</i>, Fab genes was not mutated, meaning that bacteria can synthesis fatty acids. Therefore, there may be a basal level fatty acids concentration inside the cell, making the transcription not being totally repressed. </p> |
| − | <p align="justify"> | + | <p align="justify">For more details, please visit our <a href="http://2012.igem.org/Team:WHU-China/Project?catalog=2">wiki</a>.</p> |
</html> | </html> | ||
Latest revision as of 03:51, 18 October 2014
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.
Experiment
We investigated FadR expression using a Tecan Plate Reader. LB broth containing varying concentrations of oleic acid were innoculated with DH5a E. coli cells expressing fadR (BBa_K861060), fadR-RFP (BBa_K861060), or consituative RFP (BBa_J23110). These were loaded into a clear flat bottomed black walled 96 well plate, some wells contained no cells and were used as blanks. Cells were grown in the Tecan plate reader overnight at 37oC with shaking for a period of 16 hours. OD600 measurements were taken every 30 minutes. In addition RFP readings were also taken with 565nm excitation and 605nm emission wavelengths.
Values were normalised by OD and are shown as a mean of four repeats.
In the graphs above, a trend can be seen for all concentrations of oleic acid, that fluorescence produced from constitutively expressed RFP increases over time. In comparison there was no observable trend in fadR-RFP expressing cells, showing no increase in fluorescence in any oleic acid concentration. In addition there was no difference in the amount of fluorescence observed between the fadR-RFP and the fadR only and empty cells.
The FadR promoter and BBa_J23110 constitutive promoter upstream of RFP are the same in strength and so this disregards the observed difference in expression under the two promoters being one based on strength. Further investigation was carried out using fluorescence microscopy to measure RFP fluorescence in response to different oleic acid concentrations and a similar result was observed.
Based on this information and the results shown above, we suggest that the FadR promoter does not result in expression of RFP downstream in the presence of oleic acid.
