Team:Uppsala/InterlabStudy

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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<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><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><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.<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>';

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document.getElementById("tab3").innerHTML = '<p><a href="https://static.igem.org/mediawiki/2014/7/78/Uppsala-igem2014-ILS_Raw_Data.xls">Interlab study raw data</a></p>';

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