http://2009.igem.org/wiki/index.php?title=Team:Heidelberg/Project_Measurement&feed=atom&action=historyTeam:Heidelberg/Project Measurement - Revision history2024-03-28T11:56:30ZRevision history for this page on the wikiMediaWiki 1.16.5http://2009.igem.org/wiki/index.php?title=Team:Heidelberg/Project_Measurement&diff=159425&oldid=prevHannahMeyer: /* Real-time RT-PCR */2009-10-21T23:22:36Z<p><span class="autocomment">Real-time RT-PCR</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== Real-time RT-PCR ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== Real-time RT-PCR ===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Real-time RT-PCR, which has been developed tremendously during the last 20 years, is a specific and sensitive technique for detecting and quantification of gene expression on RNA level. In order to obtain reliable results and reduce the variance during the extraction, reverse transcription and PCR, we did the measurement for each promoter with 12 replicates from an identical sample. Prior to PCR, we extracted the total RNA using Qiagen RNeasy Plant Mini Kit following the protocol from the handbook [[Team:Heidelberg/Project_Measurement#References|[15]]]. The extraction was performed by the QIAcube<sup>TM</sup> [[Team:Heidelberg/Project_Measurement#References|[16]]], the automated spin-column kits preparation robot, to avoid possible operator error. In order to perform the real-time PCR, primers and probes were designed according to QuantiFast Probe RT-PCR Handbook [[Team:Heidelberg/Project_Measurement#References|[17]]]: The target PCR product length is set between 70-200 bp. The target spans over exon-exon boundary to exclude amplification of genome DNA which may be presented in the RNA extraction as contamination. The probes have reporter fluorophore and quencher fluorophore at 5' and 3' end respectively. During amplification, probes bind specifically to the target gene. The polymerase hydrolyzes the probe during elongation due to its exonuclease activity. The signal of the released fluorophore can then be detected and correlates therefore to the amount of PCR product. The real-time RT-PCR was performed on StepOnePlus<sup>TM</sup> Real-time PCR System from Applied Biosystems [[Team:Heidelberg/Project_Measurement#References|[18]]]. We quantified the expression of GFP, the reporter gene, together with other 5 housekeeping genes (ß-actin, glycerol-aldehyde-3-phosphate de-hydrogenase, glucose-6-phosphate dehydrogenase, gamma-tubulin, 18S rRNA) for normalization. At the end of each cycle, the fluorophore of each well is read and recorded. Ct (threshold cycle) is defined as the cycle number at which the fluorescent strength (the curve) crosses a certain threshold (Fig. 11). The more amount of starting mRNA, the sooner will the machine detect the fluorescent signals from the PCR reaction, which means a smaller Ct under the same threshold. The threshold should be a careful decision. With a too small threshold, the signal may be too weak for reliable detection. If it is too big, the reaction might be restricted by the enzymes and other materials. Usually, we set the threshold at 0.05 for all plates. Calculating the difference between Ct values, with normalization of the multiple housekeeping genes, we get the ratio of the activity of the promoter of interest and our reference.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Real-time RT-PCR, which has been developed tremendously during the last 20 years, is a specific and sensitive technique for detecting and quantification of gene expression on RNA level. In order to obtain reliable results and reduce the variance during the extraction, reverse transcription and PCR, we did the measurement for each promoter with 12 replicates from an identical sample. Prior to PCR, we extracted the total RNA using Qiagen RNeasy Plant Mini Kit following the protocol from the handbook [[Team:Heidelberg/Project_Measurement#References|[15]]]. The extraction was performed by the QIAcube<sup>TM</sup> [[Team:Heidelberg/Project_Measurement#References|[16]]], the automated spin-column kits preparation robot, to avoid possible operator error. In order to perform the real-time PCR, primers and probes were designed according to QuantiFast Probe RT-PCR Handbook [[Team:Heidelberg/Project_Measurement#References|[17]]]: The target PCR product length is set between 70-200 bp. The target spans over exon-exon boundary to exclude amplification of genome DNA which may be presented in the RNA extraction as contamination. The probes have reporter fluorophore and quencher fluorophore at 5' and 3' end respectively. During amplification, probes bind specifically to the target gene. The polymerase hydrolyzes the probe during elongation due to its exonuclease activity. The signal of the released fluorophore can then be detected and correlates therefore to the amount of PCR product. The real-time RT-PCR was performed on StepOnePlus<sup>TM</sup> Real-time PCR System from Applied Biosystems [[Team:Heidelberg/Project_Measurement#References|[18]]]. We quantified the expression of GFP, the reporter gene, together with other 5 housekeeping genes (ß-actin, glycerol-aldehyde-3-phosphate de-hydrogenase, glucose-6-phosphate dehydrogenase, gamma-tubulin, 18S rRNA) for normalization. At the end of each cycle, the fluorophore of each well is read and recorded. Ct (threshold cycle) is defined as the cycle number at which the fluorescent strength (the curve) crosses a certain threshold (Fig. 11). The more amount of starting mRNA, the sooner will the machine detect the fluorescent signals from the PCR reaction, which means a smaller Ct under the same threshold. The threshold should be a careful decision. With a too small threshold, the signal may be too weak for reliable detection. If it is too big, the reaction might be restricted by the enzymes and other materials. Usually, we set the threshold at 0.05 for all plates <ins class="diffchange diffchange-inline">(Fig. 12.1 and Fig. 12.2)</ins>. Calculating the difference between Ct values, with normalization of the multiple housekeeping genes, we get the ratio of the activity of the promoter of interest and our reference.</div></td></tr>
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</table>HannahMeyerhttp://2009.igem.org/wiki/index.php?title=Team:Heidelberg/Project_Measurement&diff=159242&oldid=prevHannahMeyer: /* Flow Cytometry */2009-10-21T23:17:11Z<p><span class="autocomment">Flow Cytometry</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="300px" style="padding: 0 20px 0 0;"|</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="300px" style="padding: 0 20px 0 0;"|</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[image:HD09_GFP_negative.png|center|300px|thumb|<div style="text-align:justify;">'''<del class="diffchange diffchange-inline">Fig. </del>9 : Example of a negative control in Hela cells by flow cytometry.''' The number of events is plotted against the fluorescence (log) of GFP. Background signal is set under 10 and the area under the curve is colored in green.</div>]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[image:HD09_GFP_negative.png|center|300px|thumb|<div style="text-align:justify;">'''<ins class="diffchange diffchange-inline">Figure </ins>9 : Example of a negative control in Hela cells by flow cytometry.''' The number of events is plotted against the fluorescence (log) of GFP. Background signal is set under 10 and the area under the curve is colored in green.</div>]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="30px"|</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="30px"|</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[image:HD09_GFP_positive_HeLa_Jet.png|center|300px|thumb| <div style="text-align:justify;">'''<del class="diffchange diffchange-inline">Fig. </del>10 : Example of a positive control (JeT) in Hela cells by flow cytometry.''' The number of events is plotted against the fluorescence (log) of GFP. The area under the curve is colored in green.</div>]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[image:HD09_GFP_positive_HeLa_Jet.png|center|300px|thumb| <div style="text-align:justify;">'''<ins class="diffchange diffchange-inline">Figure </ins>10 : Example of a positive control (JeT) in Hela cells by flow cytometry.''' The number of events is plotted against the fluorescence (log) of GFP. The area under the curve is colored in green.</div>]]</div></td></tr>
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</table>HannahMeyerhttp://2009.igem.org/wiki/index.php?title=Team:Heidelberg/Project_Measurement&diff=159215&oldid=prevHannahMeyer: /* Real-time RT-PCR */2009-10-21T23:16:32Z<p><span class="autocomment">Real-time RT-PCR</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[image:qPCR_Ct_18s_CMV.png|center|210px|thumb|<div style="text-align:justify;">'''<del class="diffchange diffchange-inline">Fig. </del>11 : Ct values of 18s rRNA with CMV promoter 20h after transfection''' Threshold is set at 0.05, the cycle number at when the amplification plot crosses the threshold is the Ct value.</div>]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[image:qPCR_Ct_18s_CMV.png|center|210px|thumb|<div style="text-align:justify;">'''<ins class="diffchange diffchange-inline">Figure </ins>11 : Ct values of 18s rRNA with CMV promoter 20h after transfection''' Threshold is set at 0.05, the cycle number at when the amplification plot crosses the threshold is the Ct value.</div>]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="30px"|</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|width="30px"|</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[image:qPCR_Ct_eGFP_CMV.png|center|210px|thumb| <div style="text-align:justify;">'''<del class="diffchange diffchange-inline">Fig. </del>12.1 : Ct values of eGFP with CMV promoter 20h after transfection''' Threshold is set at 0.05. 12 dots represent 12 RNA extractions from identical sample.</div>]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[image:qPCR_Ct_eGFP_CMV.png|center|210px|thumb| <div style="text-align:justify;">'''<ins class="diffchange diffchange-inline">Figure </ins>12.1 : Ct values of eGFP with CMV promoter 20h after transfection''' Threshold is set at 0.05. 12 dots represent 12 RNA extractions from identical sample.</div>]]</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[image:qPCR_Ct_eGFP_JeT.png|center|210px|thumb|<div style="text-align:justify;"> '''<del class="diffchange diffchange-inline">Fig. </del>12.2 : Ct values of eGFP with JeT promoter 20h after transfection''' Threshold is set at 0.05. 12 dots represent 12 RNA extractions from identical sample.</div>]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[image:qPCR_Ct_eGFP_JeT.png|center|210px|thumb|<div style="text-align:justify;"> '''<ins class="diffchange diffchange-inline">Figure </ins>12.2 : Ct values of eGFP with JeT promoter 20h after transfection''' Threshold is set at 0.05. 12 dots represent 12 RNA extractions from identical sample.</div>]]</div></td></tr>
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</table>HannahMeyerhttp://2009.igem.org/wiki/index.php?title=Team:Heidelberg/Project_Measurement&diff=159093&oldid=prevHannahMeyer: /* Different core promoters result in different expression strength */2009-10-21T23:12:36Z<p><span class="autocomment">Different core promoters result in different expression strength</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:HD09_CMV_standard3.png|thumb|left|300px|<div style="text-align:justify;">'''<del class="diffchange diffchange-inline">Fig. </del>6: Flow cytometry and microscopy measurement data of CMV (REU) in different cell lines.''' The three cell lines MCF-7, U2-OS and HeLa were cotransfected with the CMV promoter coupled to GFP and a reference plasmid including the promoter JeT coupled to mCherry. The relative fluorescence (REU) of GFP was measured 20 hours after transfection. All cell lines were measured once for microscopy. The HeLa cell line was measured five times by flow cytometry and MCF-7 and U2-OS were measured four times. In the flow cytometry measurement the standard error of the mean (SEM) is indicated by the error bars.</div> ]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:HD09_CMV_standard3.png|thumb|left|300px|<div style="text-align:justify;">'''<ins class="diffchange diffchange-inline">Figure </ins>6: Flow cytometry and microscopy measurement data of CMV (REU) in different cell lines.''' The three cell lines MCF-7, U2-OS and HeLa were cotransfected with the CMV promoter coupled to GFP and a reference plasmid including the promoter JeT coupled to mCherry. The relative fluorescence (REU) of GFP was measured 20 hours after transfection. All cell lines were measured once for microscopy. The HeLa cell line was measured five times by flow cytometry and MCF-7 and U2-OS were measured four times. In the flow cytometry measurement the standard error of the mean (SEM) is indicated by the error bars.</div> ]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:JeT_CMV_standard2.png|thumb|left|300px|<div style="text-align:justify;">'''<del class="diffchange diffchange-inline">Fig. </del>7: Flow cytometry measurement data of JeT_CMV (REU) in different cell lines.''' The three cell lines MCF-7, U2-OS and HeLa were cotransfected with the JeT_CMV promoter coupled to GFP and a reference plasmid including the promoter JeT coupled to mCherry. The relative fluorescence (REU) of GFP was measured 20 hours after transfection. The MCF-7 and the U2-OS cell line were measured four times and the HeLa cell line five times. The standard error of the mean (SEM) is indicated by the error bars. </div>]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:JeT_CMV_standard2.png|thumb|left|300px|<div style="text-align:justify;">'''<ins class="diffchange diffchange-inline">Figure </ins>7: Flow cytometry measurement data of JeT_CMV (REU) in different cell lines.''' The three cell lines MCF-7, U2-OS and HeLa were cotransfected with the JeT_CMV promoter coupled to GFP and a reference plasmid including the promoter JeT coupled to mCherry. The relative fluorescence (REU) of GFP was measured 20 hours after transfection. The MCF-7 and the U2-OS cell line were measured four times and the HeLa cell line five times. The standard error of the mean (SEM) is indicated by the error bars. </div>]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Team:Heidelberg/Project_Measurement#Measurement|[TOP]]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Team:Heidelberg/Project_Measurement#Measurement|[TOP]]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== A stable cell line for promoter measurement ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== A stable cell line for promoter measurement ===</div></td></tr>
</table>HannahMeyerhttp://2009.igem.org/wiki/index.php?title=Team:Heidelberg/Project_Measurement&diff=159085&oldid=prevHannahMeyer: /* Measuring RMPU by real-time RT-PCR */2009-10-21T23:12:10Z<p><span class="autocomment">Measuring RMPU by real-time RT-PCR</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-valign="top" border="0"</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-valign="top" border="0"</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>|[[Image:QPCR_result_matlab.png| thumb |left|300px|<div style="text-align:justify;">'''<del class="diffchange diffchange-inline">Fig. </del>4: Real-time RT-PCR data of CMV and JeT promoters.''' One group of HeLa cells were transfected with plasmid containing CMV promoter coupled to GFP. Another group with JeT promoter coupled to GFP was used as reference. RNA was extracted after 20 h and 50 h, followed by real-time RT-PCR. The Ct values were collected with a threshold of 0.05. The CMV activity compared to JeT at the same time point was calculated in MatLab as "arbitrary units" which correspond to amount of mRNA.</div> ]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|[[Image:QPCR_result_matlab.png| thumb |left|300px|<div style="text-align:justify;">'''<ins class="diffchange diffchange-inline">Figure </ins>4: Real-time RT-PCR data of CMV and JeT promoters.''' One group of HeLa cells were transfected with plasmid containing CMV promoter coupled to GFP. Another group with JeT promoter coupled to GFP was used as reference. RNA was extracted after 20 h and 50 h, followed by real-time RT-PCR. The Ct values were collected with a threshold of 0.05. The CMV activity compared to JeT at the same time point was calculated in MatLab as "arbitrary units" which correspond to amount of mRNA.</div> ]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>|[[Image:qpcr_result.png| thumb |left|300px|<div style="text-align:justify;">'''<del class="diffchange diffchange-inline">Fig. </del>5: Real-time RT-PCR data of CMV promoter'''. Arbitrary units of CMV divided by that of JeT is the [https://2009.igem.org/Team:Heidelberg/Project_Measurement RMPU]. </div>]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|[[Image:qpcr_result.png| thumb |left|300px|<div style="text-align:justify;">'''<ins class="diffchange diffchange-inline">Figure </ins>5: Real-time RT-PCR data of CMV promoter'''. Arbitrary units of CMV divided by that of JeT is the [https://2009.igem.org/Team:Heidelberg/Project_Measurement RMPU]. </div>]]</div></td></tr>
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</table>HannahMeyerhttp://2009.igem.org/wiki/index.php?title=Team:Heidelberg/Project_Measurement&diff=159076&oldid=prevHannahMeyer: /* Measuring RMPU by real-time RT-PCR */2009-10-21T23:11:27Z<p><span class="autocomment">Measuring RMPU by real-time RT-PCR</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== Measuring RMPU by real-time RT-PCR ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== Measuring RMPU by real-time RT-PCR ===</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>To measure the RMPU (Relative Mammalian Promoter Units), HeLa cells were transfected with <del class="diffchange diffchange-inline">plasmid </del>containing the promoter of interest. HeLa cells transfected with plasmid containing JeT promoter were used as reference. <del class="diffchange diffchange-inline">The transcription efficiency was checked using flow cytometry. After </del>20 h and 50 h <del class="diffchange diffchange-inline">expression</del>, total RNA (> 200 bp) was isolated<del class="diffchange diffchange-inline">, </del>followed by <del class="diffchange diffchange-inline">the </del>real-time RT-PCR, where the mRNA amount of GFP was quantified. For each promoter at each time point, 12 replicates were taken to obtain reliable results and reduce the variance. To get comparable results of different samples, we used multiple housekeeping genes as internal <del class="diffchange diffchange-inline">control</del>, while using non-transfected HeLa mRNA as plate-to-plate correction. <!--The program was performed with ABI StepOnePlus Real-time PCR System. Data was collected from the associated software. GFP expression was normalized against housekeeping genes using own written MatLab-script.--> The CMV promoter was calculated about 2.89 times <del class="diffchange diffchange-inline">as strong </del>as JeT after 20 h and 2.04 times after 50 h (Fig. 4 and 5).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>To measure the RMPU (Relative Mammalian Promoter Units), HeLa cells were transfected with <ins class="diffchange diffchange-inline">plasmids </ins>containing the promoter of interest. HeLa cells transfected with plasmid containing JeT promoter were used as reference. <ins class="diffchange diffchange-inline">At two different time-points (</ins>20 h and 50 h <ins class="diffchange diffchange-inline">after transfection)</ins>, total RNA (> 200 bp) was isolated<ins class="diffchange diffchange-inline">. This step was </ins>followed by <ins class="diffchange diffchange-inline"> </ins>real-time RT-PCR, where the mRNA amount of GFP was quantified. For each promoter at each time point, 12 replicates were taken to obtain reliable results and reduce the variance. To get comparable results of different samples, we used multiple housekeeping genes as internal <ins class="diffchange diffchange-inline">controls</ins>, while using non-transfected HeLa mRNA as plate-to-plate correction. <!--The program was performed with ABI StepOnePlus Real-time PCR System. Data was collected from the associated software. GFP expression was normalized against housekeeping genes using own written MatLab-script.--> The CMV promoter was calculated about 2.89 times <ins class="diffchange diffchange-inline">stronger </ins>as JeT after 20 h and 2.04 times <ins class="diffchange diffchange-inline">stronger </ins>after 50 h (Fig. 4 and 5).</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Team:Heidelberg/Project_Measurement#Measurement|[TOP]]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Team:Heidelberg/Project_Measurement#Measurement|[TOP]]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== Measuring REU by flow cytometry and image analysis===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== Measuring REU by flow cytometry and image analysis===</div></td></tr>
</table>HannahMeyerhttp://2009.igem.org/wiki/index.php?title=Team:Heidelberg/Project_Measurement&diff=158872&oldid=prevHannahMeyer: /* Variety of cell lines */2009-10-21T23:05:06Z<p><span class="autocomment">Variety of cell lines</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <div style="text-align:justify;"> TOP10 or DH5&alpha; cells have been widely accepted as chassis systems by synthetic biologists working in bacteria. For the work with mammalian cells, no such consensus exists; also, it would not be sensible to limit synthetic mammalian biology to a small number of cell lines, as every cell line is suited for a special application. Scientists working on breast cancer virotherapy by synthetic promoters would choose a breast cancer cell line, whereas scientists working on [https://2008.igem.org/Team:Bay_Area_RSI stem cell therapies to myocardial infarction] would choose a cardiomyocyte cell line. Cell lines differ greatly, even in expression strength of constitutive promoters (shown [[Team:Heidelberg/Project_Measurement#Measuring_REU_by_flow_cytometry_and_image_analysis|below]]).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <div style="text-align:justify;"> TOP10 or DH5&alpha; cells have been widely accepted as chassis systems by synthetic biologists working in bacteria. For the work with mammalian cells, no such consensus exists; also, it would not be sensible to limit synthetic mammalian biology to a small number of cell lines, as every cell line is suited for a special application. Scientists working on breast cancer virotherapy by synthetic promoters would choose a breast cancer cell line, whereas scientists working on [https://2008.igem.org/Team:Bay_Area_RSI stem cell therapies to myocardial infarction] would choose a cardiomyocyte cell line. Cell lines differ greatly, even in expression strength of constitutive promoters (shown [[Team:Heidelberg/Project_Measurement#Measuring_REU_by_flow_cytometry_and_image_analysis|below]]).</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <div style="text-align:justify;">Each part must be characterized in every cell line. We worked with three cancer cell lines, [[Team:Heidelberg/Eukaryopedia#HeLa|HeLa]] (cervical cancer), [[Team:Heidelberg/Eukaryopedia#MCF-7|MCF-7]] (breast cancer) and [[Team:Heidelberg/Eukaryopedia#U2-OS|U2-OS]] (osteosarcoma). We suggest that the synthetic biology community should pick a small number of cell lines as model systems. We argue that HeLa (well known, widely used, easy to transfect) and/or MCF-7 (very robust to apoptosis) should be part of these cell lines. </div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <div style="text-align:justify;">Each part must be characterized in every cell line. We worked with three cancer cell lines, [[Team:Heidelberg/Eukaryopedia#HeLa|HeLa]] (cervical cancer), [[Team:Heidelberg/Eukaryopedia#MCF-7|MCF-7]] (breast cancer) and [[Team:Heidelberg/Eukaryopedia#U2-OS|U2-OS]] (osteosarcoma). We suggest that the synthetic biology community should pick a small number of cell lines as model systems. We argue that HeLa (well known, widely used, easy to transfect) and/or MCF-7 (very robust to apoptosis) should be part of these cell lines. </div></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== Lack of truly constitutive promoters in mammalian cells ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== Lack of truly constitutive promoters in mammalian cells ====</div></td></tr>
</table>HannahMeyerhttp://2009.igem.org/wiki/index.php?title=Team:Heidelberg/Project_Measurement&diff=158723&oldid=prevMichaelBartoschek at 23:01, 21 October 20092009-10-21T23:01:17Z<p></p>
<a href="http://2009.igem.org/wiki/index.php?title=Team:Heidelberg/Project_Measurement&diff=158723&oldid=158577">Show changes</a>MichaelBartoschekhttp://2009.igem.org/wiki/index.php?title=Team:Heidelberg/Project_Measurement&diff=158577&oldid=prevMichaelBartoschek at 22:57, 21 October 20092009-10-21T22:57:11Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>However, a common problem when measuring any construct or device in mammalian cells is the fact that the transfection rate is not always constant. Thus, one never knows how many copies of a construct are actually in the cell. This is why we attempted to create a system that allows a controlled integration of our constructs into the genome. To accomplish this we used the FRT/Flp system which is based on homologous recombination by the enzyme flippase at a specific sequence – the FRT site [[Team:Heidelberg/Project_Measurement#References|[20]]]. [[Team:Heidelberg/stables|Read more about our stable cell lines]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>However, a common problem when measuring any construct or device in mammalian cells is the fact that the transfection rate is not always constant. Thus, one never knows how many copies of a construct are actually in the cell. This is why we attempted to create a system that allows a controlled integration of our constructs into the genome. To accomplish this we used the FRT/Flp system which is based on homologous recombination by the enzyme flippase at a specific sequence – the FRT site [[Team:Heidelberg/Project_Measurement#References|[20]]]. [[Team:Heidelberg/stables|Read more about our stable cell lines]]</div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[[Team:Heidelberg/Project_Measurement#Measurement|[TOP]]]</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Results ==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Results ==</div></td></tr>
</table>MichaelBartoschekhttp://2009.igem.org/wiki/index.php?title=Team:Heidelberg/Project_Measurement&diff=155962&oldid=prevHannahMeyer: /* Lack of truly constitutive promoters in mammalian cells */2009-10-21T21:34:12Z<p><span class="autocomment">Lack of truly constitutive promoters in mammalian cells</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:HD09_FACS_cmv_medium_with_time.PNG|thumb|left|300px| <div style="text-align:justify;">'''Fig. 2: CMV and JeT strength changes depending on conditions''' We characterize GFP expression from [http://partsregistry.org/wiki/index.php?title=Part:BBa_K203112 JeT] and [http://partsregistry.org/Part:BBa_I712004 CMV] fluctuate dependent on condition (Everolimus induces extreme starvation). Measured by flow cytometry 20 hours after transfection (unless specified otherwise) in MCF-7. The standard deviation is represented by the error bars. </div>]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:HD09_FACS_cmv_medium_with_time.PNG|thumb|left|300px| <div style="text-align:justify;">'''Fig. 2: CMV and JeT strength changes depending on conditions''' We characterize GFP expression from [http://partsregistry.org/wiki/index.php?title=Part:BBa_K203112 JeT] and [http://partsregistry.org/Part:BBa_I712004 CMV] fluctuate dependent on condition (Everolimus induces extreme starvation). Measured by flow cytometry 20 hours after transfection (unless specified otherwise) in MCF-7. The standard deviation is represented by the error bars. </div>]]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Figure 2 shows measurements of GFP expression from [http://partsregistry.org/Part:BBa_I712004 CMV] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K203112 JeT] under varying conditions. This result demonstrates that every promoter in mammalian cells underlies regulation, and therefore, is not truly constitutive. We analyzed the sequence of CMV by [http://www.gene-regulation.com/pub/databases.html TRANSFAC Professional] and found it to contain two [[Team:Heidelberg/Eukaryopedia#Transcription_factors|NF-&kappa;B]] binding sites, two [[Team:Heidelberg/Eukaryopedia#CREB|CREB]]-binding sites, and single [[Team:Heidelberg/Eukaryopedia#Ap1|Ap1]], RFX1 and SRF binding sites. Of NF-&kappa;B, CREB and Ap1, we know that they have a high constitutive activity ([[Team:Heidelberg/Project_Synthetic_promoters#Generation_of_a_library_of_constitutive_promoters|compare to Synthetic promoters]]), but nevertheless, they underlie regulation. For example, NF-&kappa;B is induced by inflammation conditions, whereas CREB is activated by the second messenger cAMP[[Team:Heidelberg/Project_Measurement#References|[8]]] and thus responds to many hormones, starvation conditions etc. This impedes comparison of promoters in different conditions. We next [[Team:Heidelberg/Project_Measurement#Characterization_of_promoters_under_different_conditions|discuss]] how this can be achieved.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Figure 2 shows measurements of GFP expression from [http://partsregistry.org/Part:BBa_I712004 CMV] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K203112 JeT] under varying conditions. This result demonstrates that every promoter in mammalian cells underlies regulation, and therefore, is not truly constitutive. We analyzed the sequence of CMV by [http://www.gene-regulation.com/pub/databases.html TRANSFAC Professional] and found it to contain two [[Team:Heidelberg/Eukaryopedia#Transcription_factors|NF-&kappa;B]] binding sites, two [[Team:Heidelberg/Eukaryopedia#CREB|CREB]]-binding sites, and single [[Team:Heidelberg/Eukaryopedia#Ap1|Ap1]], RFX1 and SRF binding sites. Of NF-&kappa;B, CREB and Ap1, we know that they have a high constitutive activity ([[Team:Heidelberg/Project_Synthetic_promoters#Generation_of_a_library_of_constitutive_promoters|compare to Synthetic promoters]]), but nevertheless, they underlie regulation. For example, NF-&kappa;B is induced by inflammation conditions, whereas CREB is activated by the second messenger cAMP[[Team:Heidelberg/Project_Measurement#References|[8]]] and thus responds to many hormones, starvation conditions etc. This impedes comparison of promoters in different conditions. We next [[Team:Heidelberg/Project_Measurement#Characterization_of_promoters_under_different_conditions|discuss]] how this can be achieved.</div></td></tr>
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</table>HannahMeyer