Team:Uppsala/InterlabStudy

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document.getElementById("tab2").innerHTML = '<h2>INTRO</h2><p>The plan was to do the Interlab Study for all Andersson promoters and test both obligatory constructs with BFP as reporter. However there were several problems and this made us think about how the study could be improved.</p><h2>PROBLEMS</h2><p>When we did our first measurements we discovered two things, all BFP constructs efficiently killed our cells (2% of population fluorescent) the same went for our stronger Andersson promoters with GFP. We were told by our advisor that this was common for measurements done in high copy plasmids. There are two possible reasons why, either the protein is expressed in a toxic amount or the investment in protein expression is so encumbering that any cells that lose this expression out-competes the correct cells.<br><br>A solution to this problem would be to use a low copy plasmid. This would lower the stress for the cells both by reducing the amount of plasmid present and the amount of protein expressed.<br><br>There is a small problem with this though. As can be seen in figure 1 the amount of noise in our measurements is already rather high, after some research we discovered that the GFP used for this study is horribly outdated and weak compared to other fluorescent reporters available.</p><h2>SUGGESTIONS</h2><p>We believe that this study is an admirable effort to improve measurement consistency and standard data for the most common promoters. However we would like to suggest the following changes</p><br><br><ul><li>1) Use a stronger reporter: YFP is included in the kit and has been proven to be more efficient, Superfolder GFP has been available since 2006 and should have been  included in the kit by now. A stronger reporter reduces the effect of noise and allows for studies in lower copy plasmids</li><li>2) Use a low copy plasmid (pSB3K3 has worked well for us): This reduces cell stress. </li><li>3) The difference between the ribosome binding sites B0032 and B0034 should be studied further and B0034 should be used as a standard for future measurements since it is the most commonly used.</li></ul>';
document.getElementById("tab2").innerHTML = '<h2>INTRO</h2><p>The plan was to do the Interlab Study for all Andersson promoters and test both obligatory constructs with BFP as reporter. However there were several problems and this made us think about how the study could be improved.</p><h2>PROBLEMS</h2><p>When we did our first measurements we discovered two things, all BFP constructs efficiently killed our cells (2% of population fluorescent) the same went for our stronger Andersson promoters with GFP. We were told by our advisor that this was common for measurements done in high copy plasmids. There are two possible reasons why, either the protein is expressed in a toxic amount or the investment in protein expression is so encumbering that any cells that lose this expression out-competes the correct cells.<br><br>A solution to this problem would be to use a low copy plasmid. This would lower the stress for the cells both by reducing the amount of plasmid present and the amount of protein expressed.<br><br>There is a small problem with this though. As can be seen in figure 1 the amount of noise in our measurements is already rather high, after some research we discovered that the GFP used for this study is horribly outdated and weak compared to other fluorescent reporters available.</p><h2>SUGGESTIONS</h2><p>We believe that this study is an admirable effort to improve measurement consistency and standard data for the most common promoters. However we would like to suggest the following changes</p><br><br><ul><li>1) Use a stronger reporter: YFP is included in the kit and has been proven to be more efficient, Superfolder GFP has been available since 2006 and should have been  included in the kit by now. A stronger reporter reduces the effect of noise and allows for studies in lower copy plasmids</li><li>2) Use a low copy plasmid (pSB3K3 has worked well for us): This reduces cell stress. </li><li>3) The difference between the ribosome binding sites B0032 and B0034 should be studied further and B0034 should be used as a standard for future measurements since it is the most commonly used.</li></ul>';
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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.  <br><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/2014/9/9e/Uppsala_igem2014_ILS_Raw_Data_%281%29.pdf">Interlab study raw data.pdf</a>';
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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.  <br><br><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>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/2014/9/9e/Uppsala_igem2014_ILS_Raw_Data_%281%29.pdf">Interlab study raw data.pdf</a>';
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