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| | <div class="row"> | | <div class="row"> |
| | <div class="twelve columns centered text-center"> | | <div class="twelve columns centered text-center"> |
| − | <h1>Interlab<span>.</span></h1> | + | <h1>Interlab</h1> |
| − | <p class="title1" style="text-align:center">The Peking iGEM 2016 team is participating in the third year of the iGEM Interlab study along with about 100 teams.We focused on quantify expression of GFP in common, comparable or absolute units. </p> | + | <p class="title1" style="text-align:center">The Peking iGEM 2016 team is participating in the third year of the iGEM Interlab study along with about 100 teams. We focused on quantifying expression of GFP in common, comparable or absolute units. </p> |
| | </div> | | </div> |
| | </div> | | </div> |
| | </div> | | </div> |
| | <!-- Page Title End--> | | <!-- Page Title End--> |
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| | <style type="text/css"> | | <style type="text/css"> |
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| | + | color: #11abb0; |
| | + | font-size: 38px; |
| | + | line-height: 48px; |
| | + | margin-bottom: 12px; |
| | + | font-family: raleway-bold, sans-serif; |
| | + | background: transparent; |
| | + | letter-spacing: 3px; |
| | + | text-transform: uppercase; |
| | + | font-weight: 350; |
| | + | text-align: center; |
| | + | } |
| | #primary span{ | | #primary span{ |
| | display:block; | | display:block; |
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| | border-bottom: 1px solid #eee; | | border-bottom: 1px solid #eee; |
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| | menuFixed('sidebar'); | | menuFixed('sidebar'); |
| | } | | } |
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| | <li><a href="#protocol">Protocol</a></li> | | <li><a href="#protocol">Protocol</a></li> |
| | </ul> | | </ul> |
| | + | <h4>Description</h4> |
| | <h4>Results</h4> | | <h4>Results</h4> |
| − | | + | |
| | <li><a href="#sequencing">Sequencing</a></li> | | <li><a href="#sequencing">Sequencing</a></li> |
| | <li><a href="#data">Data</a></li> | | <li><a href="#data">Data</a></li> |
| | </ul> | | </ul> |
| − | </ul>
| + | <h4>Discussion</h4> |
| | </div> | | </div> |
| | </div> | | </div> |
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| | </div> | | </div> |
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| − | <div class="nine columns"> | + | <div class="nine columns texttext"> |
| − | | + | <div class="texttitle">Methods</div> |
| | <div> | | <div> |
| | <a id="background"></a> | | <a id="background"></a> |
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| | <a id="design"></a> | | <a id="design"></a> |
| | <h3 class="classic-title";"><span>Design</span></h3> | | <h3 class="classic-title";"><span>Design</span></h3> |
| − | <p>Fluorescence is widely used as a proxy for promoter activity by expressing fluorescent proteins such as green fluorescent protein (GFP). While this is an indirect measurement, it provides a useful insight into expression levels and has the significant advantage that it can be monitored continuously without disrupting cells. | + | <p>Fluorescence is widely used as a proxy for promoter activity by expressing fluorescent proteins such as green fluorescent protein (GFP). Despite this is an indirect measurement, it provides a useful insight into expression levels and has significant advantage that it could be ontinuously monitored without disrupting cells. |
| | </p> | | </p> |
| | <p>Fluorescence/OD600 is routinely used to give an adjustment of the relative expression per cell.</p> | | <p>Fluorescence/OD600 is routinely used to give an adjustment of the relative expression per cell.</p> |
| − | <p>We aim to do this using the supplied FITC as a standard reference material. You will measure the fluorescence of your instrument using a dilution series of this reference material to construct a standard curve. We have previously performed this standard curve on our own instrument alongside a standard curve for purified GFP. Using these standard curves alongside your own standard curve for FITC it is thus possible to transform your relative measurements of fluorescence into absolute measurements of GFP molecules.</p> | + | <p>We aim to do this using the supplied FITC as a standard reference material. The standard curve could be constructed via measuring the fluorescence of a dilution series. We have previously performed this standard curve on our own instrument alongside a standard curve for purified GFP. Using these standard curves alongside your own standard curve for FITC it is thus possible to transform your relative measurements of fluorescence into absolute measurements of GFP molecules.</p> |
| − | <p>However, we aim to control for instrument variability, at least to some degree, by measuring a standard scattering solution of a mono-dispersed silica suspension (LUDOX). The objective is to see if a simple, single fixed-point measurement can be used as a ratiometric adjustment to provide greater uniformity in fluorescence/OD600 measurements across sites.</p> | + | <p>However, we aim to contain instrument variability, at least to some degree, by measuring a standard scattering solution of a mono-dispersed silica suspension (LUDOX). The objective is to see if a simple, single fixed-point measurement can be used as a ratiometric adjustment to provide greater uniformity in fluorescence/OD600 measurements across sites.</p> |
| | </div> | | </div> |
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| | <a id="materials"></a> | | <a id="materials"></a> |
| | <h3 class="classic-title";"><span>Materials and methods</span></h3> | | <h3 class="classic-title";"><span>Materials and methods</span></h3> |
| − | <h5>Used plasmids</h5> | + | <h5>Plasmids used</h5> |
| | <p style="margin:0 0 0 0">• Plasmid DNA (100 pg/uL in 10uL of Buffer EB)</p> | | <p style="margin:0 0 0 0">• Plasmid DNA (100 pg/uL in 10uL of Buffer EB)</p> |
| | <div style="padding-left:20px"> | | <div style="padding-left:20px"> |
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| | o Test Device 2: J23106.B0034.E0040.B0015 in pSB1C3<br> | | o Test Device 2: J23106.B0034.E0040.B0015 in pSB1C3<br> |
| | o Test Device 3: J23117.B0034.E0040.B0015 in pSB1C3<br> | | o Test Device 3: J23117.B0034.E0040.B0015 in pSB1C3<br> |
| − | o Positive Control Device: I20270 in pSB1C3 Also located in Kit Plate 3, well 8P<br> | + | o Positive Control Device: I20270 in pSB1C3 (Also located in Kit Plate 3, well 8P)<br> |
| − | o Negative Control Device: R0040 in pSB1C3 Also located in Kit Plate 2, well 6F<br> | + | o Negative Control Device: R0040 in pSB1C3 (Also located in Kit Plate 2, well 6F)<br> |
| | </p> | | </p> |
| | </div> | | </div> |
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| − | <h5>Used strain</h5> | + | <h5>Strain used</h5> |
| | <p>• <i>Escherichia coli</i> TOP10</p> | | <p>• <i>Escherichia coli</i> TOP10</p> |
| | | | |
| − | <h5>Used material</h5> | + | <h5>Materials</h5> |
| | <p> | | <p> |
| | • FITC Standard: one tube with dried down FITC for creating a FITC standard<br> | | • FITC Standard: one tube with dried down FITC for creating a FITC standard<br> |
| | • LUDOX: one tube with 30% colloidal silica suspended in 1mL of water<br> | | • LUDOX: one tube with 30% colloidal silica suspended in 1mL of water<br> |
| | • 1xPBS (phosphate buffered saline)<br> | | • 1xPBS (phosphate buffered saline)<br> |
| − | • Terrific broth (at half strength: 0.5x TB) or can use LB (Luria Bertani) media as an alternative<br> | + | • LB (Luria Bertani) media<br> |
| | • Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)<br> | | • Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)<br> |
| | • 50 ml Falcon tube (or equivalent) or 250 ml shake flask for cell growth<br> | | • 50 ml Falcon tube (or equivalent) or 250 ml shake flask for cell growth<br> |
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| | </p> | | </p> |
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| − | <h5>Used machines</h5> | + | <h5>Machines</h5> |
| | <p> | | <p> |
| | • Thermo VARIOSKAN FLASH<br> | | • Thermo VARIOSKAN FLASH<br> |
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| | </p> | | </p> |
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| − | <h5>Used software</h5> | + | <h5>Software</h5> |
| | <p> | | <p> |
| | • Microsoft Excel<br> | | • Microsoft Excel<br> |
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| | </p> | | </p> |
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| − | <h5>Used methods</h5> | + | <h5>Methods</h5> |
| | <p style="margin:0 0 0 0">• Calibration</p> | | <p style="margin:0 0 0 0">• Calibration</p> |
| | <div style="padding-left:45px"> | | <div style="padding-left:45px"> |
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| − | <h3 class="classic-title";"><span>Description</span></h3> | + | <div class="texttitle">Description</div> |
| − | <img src="https://static.igem.org/mediawiki/2016/9/97/T--Peking--image_interlab_1.jpg"> | + | <img src="https://static.igem.org/mediawiki/2016/9/97/T--Peking--image_interlab_1.jpg"> |
| − | <p style="text-align:center;"><small><legend>Figure 1. </legend>From left to right：D1, D2, D3, Negative, Positive.</small></p> | + | <br> |
| | <img src="https://static.igem.org/mediawiki/2016/a/ab/T--Peking--image_interlab_2.jpg"> | | <img src="https://static.igem.org/mediawiki/2016/a/ab/T--Peking--image_interlab_2.jpg"> |
| − | <p>Plasmids containing promoters and GFP were taken from The 2016 DNA Distribution Kit and all devices were transformed into E.coli. Fluorescence of colonies was checked up under UV light. </p> | + | <p style="text-align:center;"><b>Figure 1. Above: from left to right: D1, D2, D3, Negative, Positive. Below: five mediums containing five different bacteria.</b></p> |
| | + | <br> |
| | + | <p>Plasmids containing promoters and GFP were taken from The 2016 DNA Distribution Kit and all devices were transformed into <i>E. coli</i>. Fluorescence of colonies was checked up under UV light. </p> |
| | <p>5 ml of liquid LB-M medium with chloramphenicol were inoculated with two chosen colonies of each device. Liquid cultures were incubated for 16~18 hours in HONOUR INCURATOR SHAKER placed in incubator. OD of these cultures was measured by MAPADA UV-3100PC SPECTROPHOTOMETER and diluted to 0.02. Fluorescence of biological and also technical replicates was measured using Thermo VARIOSKAN FLASH following our protocol. | | <p>5 ml of liquid LB-M medium with chloramphenicol were inoculated with two chosen colonies of each device. Liquid cultures were incubated for 16~18 hours in HONOUR INCURATOR SHAKER placed in incubator. OD of these cultures was measured by MAPADA UV-3100PC SPECTROPHOTOMETER and diluted to 0.02. Fluorescence of biological and also technical replicates was measured using Thermo VARIOSKAN FLASH following our protocol. |
| | </p> | | </p> |
| − |
| |
| − | <h3 class="classic-title";"><span>Results</span></h3>
| |
| | <a id="sequencing"></a> | | <a id="sequencing"></a> |
| − | <h5>Sequencing</h5> | + | <br> |
| | + | <div class="texttitle">Results</div> |
| | + | <h3 class="classic-title";"><span>Sequencing</span></h3> |
| | <p> • Device 1: J23101+I13504<br> | | <p> • Device 1: J23101+I13504<br> |
| | • Device 2: J23106+I13504<br> | | • Device 2: J23106+I13504<br> |
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| | <a id="data"></a> | | <a id="data"></a> |
| − | <h5>Data</h5>
| + | <h3 class="classic-title";"><span>Data</span></h3> |
| − | <p><b>2.1 OD600 Reference Point</b></p> | + | <h4>OD600 Reference Point</h4> |
| − | <div style="text-align:center;width:100%;">
| + | <p><b>Table 1. OD600 Reference Point.</b></p> |
| − | <p style="text-align:center;"><small><legend>Table 1.</legend>OD600 Reference Point.</small></p> | + | <img src="https://static.igem.org/mediawiki/2016/f/f4/T--Peking--image_interlab_3.png"> |
| − | <img style="text-align:center;" src="https://static.igem.org/mediawiki/2016/f/f4/T--Peking--image_interlab_3.png"> | + | <br> |
| − | </div>
| + | <h4>FITC Standard Curve</h4> |
| − | <p><b>2.2 FITC Standard Curve</b></p> | + | <p ><b>Table 2. Data of FITC standard curve.</b></p> |
| − | <p ><small><b>Table 2. </b>Data of FITC standard curve.</small></p> | + | |
| | <img src="https://static.igem.org/mediawiki/2016/1/1b/T--Peking--image_interlab_4.png"> | | <img src="https://static.igem.org/mediawiki/2016/1/1b/T--Peking--image_interlab_4.png"> |
| | <img src="https://static.igem.org/mediawiki/2016/0/03/T--Peking--image_interlab_5.png"> | | <img src="https://static.igem.org/mediawiki/2016/0/03/T--Peking--image_interlab_5.png"> |
| − | <p ><small><b>Figure 2. </b>FITC standard curve.</small></p> | + | <p ><b>Figure 2. FITC standard curve.</b></p> |
| − | <p><b>2.3 Normalisation</b></p>
| + | <br> |
| − | <p ><small><b>Table 3. </b>Normalisation.</small></p> | + | <h4>Normalization</h4> |
| | + | <p ><b>Table 3. Normalization.</b></p> |
| | <img src="https://static.igem.org/mediawiki/2016/d/db/T--Peking--image_interlab_6.png"> | | <img src="https://static.igem.org/mediawiki/2016/d/db/T--Peking--image_interlab_6.png"> |
| − | <p><b>2.4 Cell Measurement</b></p>
| + | <br> |
| − | <p ><small><b>Table 4. </b>Raw data of Abs600 measurement.</small></p> | + | <h4>Cell Measurement</h4> |
| | + | <p ><b>Table 4. Raw data of Abs600 measurement.</b></p> |
| | <img src="https://static.igem.org/mediawiki/2016/0/04/T--Peking--image_interlab_7.png"> | | <img src="https://static.igem.org/mediawiki/2016/0/04/T--Peking--image_interlab_7.png"> |
| − | <p ><small><b>Table 5. </b>Blank substraction and correction of Abs600 measurement.</small></p> | + | <p ><b>Table 5. Blank subtraction and correction of Abs600 measurement.</b></p> |
| − | <img src="https://static.igem.org/mediawiki/2016/e/ee/T--Peking--image_interlab_8.png"> | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | <img src="https://static.igem.org/mediawiki/2016/e/ee/T--Peking--image_interlab_8.png"> |
| | <img src="https://static.igem.org/mediawiki/2016/7/71/T--Peking--image_interlab_9.png"> | | <img src="https://static.igem.org/mediawiki/2016/7/71/T--Peking--image_interlab_9.png"> |
| − | <p ><small><b>Figure 3. </b>Blank substraction and correction of Abs600 measurement.</small></p> | + | <p ><b>Figure 3. Blank subtraction and correction of Abs600 measurement.</b></p> |
| − | <p ><small><b>Table 6. </b>Raw data of fluorescence measurement.</small></p> | + | <p ><b>Table 6. Raw data of fluorescence measurement.</b></p> |
| | <img src="https://static.igem.org/mediawiki/2016/5/52/T--Peking--image_interlab_10.png"> | | <img src="https://static.igem.org/mediawiki/2016/5/52/T--Peking--image_interlab_10.png"> |
| − | <p ><small><b>Table 7. </b>Blank substraction and correction of fluorescence measurement.</small></p> | + | <p ><b>Table 7. Blank substraction and correction of fluorescence measurement.</b></p> |
| | <img src="https://static.igem.org/mediawiki/2016/3/32/T--Peking--image_interlab_11.png"> | | <img src="https://static.igem.org/mediawiki/2016/3/32/T--Peking--image_interlab_11.png"> |
| | <img src="https://static.igem.org/mediawiki/2016/6/67/T--Peking--image_interlab_12.png"> | | <img src="https://static.igem.org/mediawiki/2016/6/67/T--Peking--image_interlab_12.png"> |
| − | <p ><small><b>Figure 4. </b>Blank substraction and correction of flurescence measurement.</small></p> | + | <p ><b>Figure 4. Blank subtraction and correction of fluorescence measurement.</b></p> |
| − | <p ><small><b>Table 8. </b>Raw data of Fl/Abs600.</small></p> | + | <p ><b>Table 8.Raw data of Fl/Abs600.</b></p> |
| | <img src="https://static.igem.org/mediawiki/2016/d/d3/T--Peking--image_interlab_13.png"> | | <img src="https://static.igem.org/mediawiki/2016/d/d3/T--Peking--image_interlab_13.png"> |
| − | <p ><small><b>Table 9. </b>Raw data of average and SD.</small></p> | + | <p ><b>Table 9. Raw data of average and SD.</b></p> |
| | <img src="https://static.igem.org/mediawiki/2016/a/a1/T--Peking--image_interlab_14.png"> | | <img src="https://static.igem.org/mediawiki/2016/a/a1/T--Peking--image_interlab_14.png"> |
| | <img src="https://static.igem.org/mediawiki/2016/0/07/T--Peking--image_interlab_15.png"> | | <img src="https://static.igem.org/mediawiki/2016/0/07/T--Peking--image_interlab_15.png"> |
| − | <p ><small><b>Figure 4. </b>Average level of devices.</small></p> | + | <p ><b>Figure 4. Average level of devices.</b></p> |
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| − | <h5>Discussion</h5> | + | <div class="texttitle">Discussion</div> |
| | <p>It is noticeable that the promoter of the Device 1 is strongest followed by the promoter of the Device 2 and Device 3.</p> | | <p>It is noticeable that the promoter of the Device 1 is strongest followed by the promoter of the Device 2 and Device 3.</p> |
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| − | </div>
| + | </div> |
| | </div> <!-- row End --> | | </div> <!-- row End --> |
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