Team:Uppsala/InterlabStudy

From 2014.igem.org

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document.getElementById("tab3").innerHTML = '<p><h2>Section I: Provenance & Release</h2><br>
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document.getElementById("tab3").innerHTML = '<p><h2>Section I: Provenance & Release</h2><br><p><b>Measurements taken by:</b>Stephanie Herman, Martin Friberg and Nils Anlind<br><b>Construction of parts:</b> Martin Friberg<br><b>Acknowledgements:</b> Erik Gullberg for support operating FACS and provided reagents, team Uppsala iGEM 2014 for valuable discussion and support.</p><br><br><b>Dates of protocols:</b><br><br>Overnight Cultures: 2014-09-17 and 2014-09-25<br>FACS: 2014-09-18 and 2014-09-26<br><br><b>Inclusion of data in publication:</b><br>Nils Anlind - Yes<br>Martin Friberg - Yes<br>Stephanie Herman - Yes</p> <h2>Section II: Protocols</h2><p>Preparation of FACS-samples: Four colonies of each construct(taken from a re-streaked colony) were grown overnight in LB(about 6mL) for 18 hours in 37 degrees celsius with 300 rpm shaking. 2,5 µl culture was put into 500 µl PBS in a FACS-tube and was left to incubate for at least 1 hour in room temperature.  <b>Manufacturer and model:</b><br>BD FACSAria IIu<br><br><b>Configurations:</b><br>FITC filter band pass 530/30, top 530 nm, 30 nm width, 480 nm blue laser. Voltage applied over the photomultiplier tube: 400 V.<br><br><b>Protocol to take measurements:</b><br>The sample tubes were loaded into the FACS and measured one at a time using BD FACSDiva™ software!<br><br> <b>Method to include or exclude each sample:</b><br><br>All samples measured was included. Individual cells that lacked fluorescence of GFP or BFP was exclude in the samples via setting a fluorescence threshold. In the cases of clear peak, the threshold was set around the peak. When there was an unclear peak, the threshold was adjusted via a negative control with no fluorescence so 1% of the negative control was included in the region.<br><br><b>Controls used:</b><br>Sterile filtered water sample was used to ensure low noise, and DH5-alpha cells without plasmids was used as non-fluorescent cell control.<br><br><b>What quantities were measured:</b><br>Fluorescence per cell.<br><br><b>Time for each set of measurements:</b> for one construct 5min (four measurements)<br><b>Cost of each set of measurements:</b> for one construct, about 5 USD.<br><b>Practical limits of quantity of samples:</b> ~24 constructs per session. The machine have a variating noise background that changes if the machine needs to be re-started. This leads to increased noise in some cases high enough that you cannot read low levels of expression.</p><h2>Section III: Measured quantities</h2><p><b>Units:</b> Fluorescence measured in AU (arbitrary units) per cell.<br><b>Equivalent in SI-units:</b> No equivalent in SI-units since measurements in absolute units was not possible. Output data depends on voltage applied over the photomultiplier tube.<br><br><b>Range of measurement:</b> Since the device measures single cells the size of the sample is only limited by the runtime, the device measures 2*104 cells/s. For this study 105 cells/sample were measured.<br><b>Significant numbers on measurement:</b> Depends upon biological variation which is hard to determine.<br><b>Precision the same in the entire range? if not, how does it differ:</b> Precision is higher at higher fluorescence since the influence of background noise is decreased.<br><b>How did you answer questions above?</b> With the help of our advisor, Erik Gullberg.<br><br><b>Instrument last calibrated:</b> 2014-09-15<br><b>How was it calibrated:</b> Software calibration using BD Cytometer Setup & Tracking Beads (#642412)</p><h2>Section IV: Measurements</h2> <p><b>See attached file:</b> FACS_ILS.<br>Sheet “Sample data” contain the raw data obtained from measurements.<br> Sheet “Construct data” contains summarized data for each construct. Geometrical mean and standard deviation of the four samples was calculated and then normalized after J23101 expression during the same FACS-run to get it in relative promoter strength.</p> <a href="https://static.igem.org/mediawiki/2014/7/78/Uppsala-igem2014-ILS_Raw_Data.xls">Interlab study raw data</a>';
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<p><b>Measurements taken by:</b>Stephanie Herman, Martin Friberg and Nils Anlind<br><b>Construction of parts:</b> Martin Friberg<br><b>Acknowledgements:</b> Erik Gullberg for support operating FACS and provided reagents, team Uppsala iGEM 2014 for valuable discussion and support.</p><br><br><b>Dates of protocols:</b><br><br>Overnight Cultures: 2014-09-17 and 2014-09-25<br>
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FACS: 2014-09-18 and 2014-09-26<br><br><b>Inclusion of data in publication:</b><br>Nils Anlind - Yes<br>Martin Friberg - Yes<br>Stephanie Herman - Yes</p> <h2>Section II: Protocols</h2><p>Preparation of FACS-samples: Four colonies of each construct(taken from a re-streaked colony) were grown overnight in LB(about 6mL) for 18 hours in 37 degrees celsius with 300 rpm shaking. 2,5 µl culture was put into 500 µl PBS in a FACS-tube and was left to incubate for at least 1 hour in room temperature.  <b>Manufacturer and model:</b><br>BD FACSAria IIu<br><br><b>Configurations:</b><br>FITC filter band pass 530/30, top 530 nm, 30 nm width, 480 nm blue laser. Voltage applied over the photomultiplier tube: 400 V.<br><br><b>Protocol to take measurements:</b><br>The sample tubes were loaded into the FACS and measured one at a time using BD FACSDiva™ software!<br><br> <b>Method to include or exclude each sample:</b><br><br>All samples measured was included. Individual cells that lacked fluorescence of GFP or BFP was exclude in the samples via setting a fluorescence threshold. In the cases of clear peak, the threshold was set around the peak. When there was an unclear peak, the threshold was adjusted via a negative control with no fluorescence so 1% of the negative control was included in the region.<br><br><b>Controls used:</b><br>Sterile filtered water sample was used to ensure low noise, and DH5-alpha cells without plasmids was used as non-fluorescent cell control.<br><br><b>What quantities were measured:</b><br>Fluorescence per cell.<br><br><b>Time for each set of measurements:</b> for one construct 5min (four measurements)<br>
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<b>Cost of each set of measurements:</b> for one construct, about 5 USD.<br><b>Practical limits of quantity of samples:</b> ~24 constructs per session. The machine have a variating noise background that changes if the machine needs to be re-started. This leads to increased noise in some cases high enough that you cannot read low levels of expression.</p><h2>Section III: Measured quantities</h2><p><b>Units:</b> Fluorescence measured in AU (arbitrary units) per cell.<br><b>Equivalent in SI-units:</b> No equivalent in SI-units since measurements in absolute units was not possible. Output data depends on voltage applied over the photomultiplier tube.<br><br><b>Range of measurement:</b> Since the device measures single cells the size of the sample is only limited by the runtime, the device measures 2*104 cells/s. For this study 105 cells/sample were measured.<br><b>Significant numbers on measurement:</b> Depends upon biological variation which is hard to determine.<br><b>Precision the same in the entire range? if not, how does it differ:</b> Precision is higher at higher fluorescence since the influence of background noise is decreased.<br><b>How did you answer questions above?</b> With the help of our advisor, Erik Gullberg.<br><br><b>Instrument last calibrated:</b> 2014-09-15<br><b>How was it calibrated:</b> Software calibration using BD Cytometer Setup & Tracking Beads (#642412)</p><h2>Section IV: Measurements</h2> <p><b>See attached file:</b> FACS_ILS.<br>Sheet “Sample data” contain the raw data obtained from measurements.<br> Sheet “Construct data” contains summarized data for each construct. Geometrical mean and standard deviation of the four samples was calculated and then normalized after J23101 expression during the same FACS-run to get it in relative promoter strength.</p> <a href="https://static.igem.org/mediawiki/2014/7/78/Uppsala-igem2014-ILS_Raw_Data.xls">Interlab study raw data</a>';
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Revision as of 12:57, 15 October 2014

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