Latest revision as of 20:41, 17 October 2014

Home

Failed to load tracking. JS is probably not enabled

Back to Top

@@ Line 5: / Line 5: @@
 document.getElementById("tab1").innerHTML = '<p><h2>The study</h2><p>“All iGEM teams are invited and encouraged to participate in the first international interlab measurement study in synthetic biology. …...The goal of the interlab study is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world.”<br><a href="https://2014.igem.org/Tracks/Measurement/Interlab_study">More info:https://2014.igem.org/Tracks/Measurement/Interlab_study</a></p><h3>Devices measured</h3><p>The following constructs were measured for the interlab study. Each construct was given a construct ID to make references more easy.<table id="partsT" style="width:100%"><tr><th>Construct ID</th><th>Construct</th></tr><tr><td>142</td><td>J23101-B0032-GFP(provided)</td></tr><tr> <td>177</td><td>J23101-B0032-GFP(constructed)</td></tr><tr><td>178</td><td>J23115-B0032-GFP</td></tr><tr><td>181</td><td>J23106-B0032-GFP</td></tr><tr><td>224</td><td>J23116-B0032-GFP</td></tr><tr><td>226</td><td>J23117-B0032-GFP</td></tr><tr><td>180</td> <td>J23105-B0032-GFP</td></tr><tr><td>187</td><td>J23101-B0034-BFP</td></tr><tr><td>188</td><td>J23115-B0034-BFP</td></tr></table><h3>Experimental detail</h3><br><br><b>Construction of devices</b><br><br><u>DNA samples from kit:</u><br><p>A DNA sample of BBa_E0240 ,BBa_J23101, BBa_J23105, BBa_J23106, BBa_K823005, BBa_J23116, BBa_J23117  were taken up from the 2014 Distribution according to “Instructions on iGEMs webpage”. They were transformed into CaCl2 DH5-alpha competent cells and plated on LB-agar plates with chloramphemnicol left to incubate at 37 degrees celsius for 18h. Two clones were re-streaked on LB-agar plates with chloramphemnicol and left to incubate for 18 hours. pSB1K3 with red-insert from 2013 years distribution was re-streaked from the -80 degree cell stock.  Single colonies were PCR-amplified with VF2 and VR primers according to protocol for “Red Taq” by “ThermoFisher Scientific”. Bands were confirmed using gel electrophoresis. An overnight culture for one verified clone for each construct was prepared. Plasmid preparations were done using “Gene elute plasmid Miniprepp” following attached protocol. Concentrations of DNA were measured using “nanodrop” at 260nm.</p><br><u>Assembly of devices:</u><p>All devices were constructed with B0032-GFP(BBa_E0240), and pSB1K3(from 2013 years distribution) combined via 3A assembly protocol, with HF restriction enzymes, cutsmart buffer and ligase provided from NEB, with each of the respective promoters to form the devices shown below. The two constructs with blue fluorescent protein (BFP) were constructed with B0034-BFP(BBa_K592024) instead of BBa_E0240. The assembled parts were transformed, overnighted and screened as explained above. The overnight cultures were stored in -80 degrees freezer in glycerol for later use in the measurements.<br><table id="partsT" style="width:100%"><tr><th>Construct</th><th>Promotor part from distribution</th></tr><tr><td>J23101-B0032-GFP</td><td>BBa_J23101</td></tr><tr> <td>J23105-B0032-GFP</td><td>BBa_J23105</td></tr><tr><td>J23106-B0032-GFP</td><td>BBa_J23106</td></tr><tr><td>J23115-B0032-GFP</td><td>BBa_K823005</td></tr><tr><td>J23116-B0032-GFP</td><td>BBa_J23116</td></tr><tr><td>J23117-B0032-GFP</td><td>BBa_J23117</td></tr><tr><td>J23101-B0034-BFP</td> <td>BBa_J23101</td></tr><tr><td>J23115-B0034-BFP</td><td>BBa_K823005</td></tr></table><h3>Preparations for measurement</h3><p>All devices were re-streaked from -80 degrees stock on LB agar plates with Kanamycin two days prior to measurement. Four separate clones were chosen from each re-streaked device and left to grow for 18 hours in 6mL of LB with Kanamycin to a final concentration of 50µg/mL. 500 µl of PBS was mixed with 2.5 µl of culture for each sample. The samples were measured after at least 1 hour using a flow cytometer.</p><h3>The measurements</h3><p>The background noise from the local environment for the FACS (BDFACSAria IIu) was checked via a water sample, and was found to be neglectable. The following settings were applied; FITC filter band pass 530/30, top 530 nm, 300nm width, 480 nm blue laser. The voltage was set to 400V over the photomultiplier tube.  Each sample was run according to manufacturers protocol.</p><h2>Result</h2><p>The results were received as a geometric mean(GM) of the four samples for each device via adjusting the fluorescence peak area to fit the peak  When no clear peak could be observed the area was adjusted via a non-fluorescent cells peak, so that max 0.5% of non-fluorescence was included in the mean . The GM of all devices were normalized to J23101-B0032-GFPs value to convert the measurement to relative promoter strength (RPU). The data from the registry was also converted to RPU in order to compare the results. The standard deviations of the samples were calculated and included as error bars in the bar plot, figure 1.</p><img src="https://static.igem.org/mediawiki/2014/7/7e/Uppsala-igem2014-Interlab1.png"><p><i>Figure 1: Promoter strengths in RPU. Blue bars are measured fluorescence, orange is registry values converted to RPU.</i></p><h2>Sequencing data</h2><p>Unfortunately there was no budget available for sequencing. Each clone 1 of the overnight culture used for the FACS-measurements was chosen to be plasmid prepped and then digested with EcoRI and PstI. 5µl of digestion together with 1µl of purple loading dye was run at 110V at gel electrophoresis, results shown in figure 2. Generuler 1kB from Thermo Fisher Scientific was used as a reference.</p><img src="https://static.igem.org/mediawiki/2014/9/93/Uppsala-igem2014-Interlab2.jpg"><p><i>Figure 2.187 and 188 show insert lengths at around 800bp. The expected length is construct( 758bp)+prefix(22bp)+suffix(21bp)=801bp for these constructs since they contain mtagBFP. All other constructs show bands at around 900-1000bp. The expected length for all GFP constructs is (919bp+prefix(22bp)+suffix(21bp)=962p. Note that construct 142 is provided in pSB3K3 which is why there is also a longer backbone-band.</i></p>';
-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 some of our stronger Andersson promoters with GFP (data not shown). 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. The problem was much more visible in our BFP construct, this is probably due to the use of the standard RBS B0034 that is much stronger than B0032 that was used for the GFP promoter tests.<br><br>A solution to the problem of large non-fluorescent subpopulations 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. 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 outdated and weak compared to other fluorescent reporters available. This could be easily corrected by the use of a more efficient reporter gene.</p><img src="https://static.igem.org/mediawiki/2014/f/fe/Uppsala-igem2014-ils.png"><p><i>Figure 1: Top graph shows GFP fluorescence for BBa_I20260, as peak to the left and as scatter plot to the right. Bottom graph shows the same for the assembled J23101-B0034-BFP in pSB1K3. Most BFP containing cells have died or mutated as can be seen in the scatter plot, only about 2% showed BFP activity. Non-fluorescent cells are shown in red.</i></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: 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 or medium copy plasmid (pSB3K3 has worked well for us): This reduces cell stress.</li><li>3. The difference between 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("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>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>';

Team:Uppsala/InterlabStudy

From 2014.igem.org

Latest revision as of 20:41, 17 October 2014

Home