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| | + | {{BNU-CHINA/partials/header}} |
| | + | {{BNU-CHINA/partials/nav | MODELING=focus}} |
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| + | <h3> Interlab </h3> |
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| + | <article id="Calibration" class="col-lg-10 col-lg-offset-1 col-md-12 col-md-offset-0 col-sm-offset-0 col-sm-12"> |
| − | <title>Proof</title>
| + | <header class="page-header"> |
| − | <meta name="viewport" content="width=device-width, initial-scale=1.0, maximum-scale=1.2, user-scalable=yes" />
| + | <h1 id="model-introduction">Calibration experiments</h1> |
| − | <meta name="description" content="Wiki of Peking iGEM 2016" />
| + | </header> |
| − | <meta name="author" content="Li Jiamian & Wang Yuqing"/>
| + | <p>Before starting cell measurements work, we accomplished three sets of calibration measurements with materials provided by the committee, including an OD600 reference point, a particle standard curve and a fluorenscein standard curve. These tests and data greatly facilitated our following quantitative interlab experiments.</p> |
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| + | <h2>Calibration 1 OD600 reference point and LUDOX</h2> |
| − | <!-- Fix Overwrite the original iGEM style=================================================== -->
| + | <p>According to the protocol, we performed three calibration experiments (OD600 reference point, particle standard curve and the fluorescence standard curve) before the body part began.</p> |
| − | <link href="https://2016.igem.org/Template:Peking/css/fix?action=raw&ctype=text/css" rel="stylesheet" />
| + | <p>In this experiment, we first uesd LUDOX CL-X provided by the committee to obtain a conversion factor as the intermediate variable between Abs600 and OD600 measurement. The LUDOX solution (45% colloidal silica suspension) is hardly scattering and gives a low absorbance value, making it an ideal material for correct the standard path length of spectrophotometers in different laboratories, which would greatly improve the accuracy of the following volume dependent experiments.</p> |
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| + | <p>Results shown below. </p> |
| − | <link href="https://2016.igem.org/Template:Peking/css/bootstrap_min?action=raw&ctype=text/css" rel="stylesheet" />
| + | <figure class="text-center"> |
| − | <link href="https://2016.igem.org/Template:Peking/css/style?action=raw&ctype=text/css" rel="stylesheet" /> | + | <img src="https://static.igem.org/mediawiki/2016/e/e9/Modeling_steps.png" alt="this is a pic" width="60%"> |
| − | <!-- CSS======================================================================================= -->
| + | <figcaption> |
| − | <link rel="stylesheet" href="https://2016.igem.org/Template:Peking/css/default?action=raw&ctype=text/css"/>
| + | Table 1. OD600 reference point |
| − | <link rel="stylesheet" href="https://2016.igem.org/Template:Peking/css/layout?action=raw&ctype=text/css"/>
| + | </figcaption> |
| − | <link rel="stylesheet" href="https://2016.igem.org/Template:Peking/css/media-queries?action=raw&ctype=text/css"/> | + | </figure> |
| − | <link rel="stylesheet" href="https://2016.igem.org/Template:Peking/css/priorstyle?action=raw&ctype=text/css"/>
| + | <p id="model-theory">4 replicates are measured respectively to acquire the average Abs600 of LUDOX CL-X solution and dH2O. Reference OD600 is given by the iGEM authority. The ratio of OD600/Abs600 is 3.652.</p> |
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| + | <h2>Calibration 2 Particle Standard Curve</h2> |
| − | color: #11abb0;
| + | <p>After standardizing the absorbance value, we managed to conclude a conversion factor between Abs600 and the estimated number of cells. In this experiment, we used the silica beads delivered by iGEM committee and made a serial dilution in the 96-well plate according to the protocol. The beads’ size and optical charateristics are similar to cells and its concentration (amount of particles per volume)is known, allowing us to conduct a standard curve of particle concentration in 4 replicates. Origin data presented at the bottom of this page.</p> |
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| + | <img src="https://static.igem.org/mediawiki/2016/e/e9/Modeling_steps.png" alt="this is a pic" width="60%"> |
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| + | <figcaption> |
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| + | Figure 1A Particle Standard Curve |
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| − | text-align: center;
| + | <figcaption> |
| − | }
| + | Figure 1B Particle Standard Curve (log scale) |
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| + | </figcaption> |
| − | .references{margin-top:150px;margin-bottom:40px;}
| + | </figure> |
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| + | <h2>Calibration 3 Fluorescence standard curve</h2> |
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| + | <p>Fluorescence value is an important synthetic biology signal, but it can vary widely in different instruments. As a result, comparing the fluorescence output of test devices by creating a standard fluorescence curve can accurately standardize the amount and fluctuation of GFP fluorescencein in provided kits. In the fluorescence curve measurement, we used the fluorescein powder delivered by committee and made a serial dilution in the 96-well plate according to the protocol. 4 replicates were measured in total. Origin data presented at the bottom of this page.</p> |
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| + | Figure 2A Fluorescein Standard Curve |
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| + | Figure 2B Fluorescein Standard Curve(log scale) |
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| − | <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>
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| − | <h4><a href="#Introduction">Introduction</a></h4>
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| − | <a id = "Introduction"></a>
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| − | <div class="texttitle">Introduction</div>
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| − | <p>The Measurement Committee is committed to achieving reliable and repeatable measurement. Therefore the interlab study is established, measurements made in different laboratories by measuring the expression of GFP will be no more variable than measurements taken within the same lab.
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| − | <p>After several years of hard work, the committee concluded a universal protocol for measuring GFP expression. In recent years, the interlab study aims to improve the protocol and identify and correct the sources of systematic variability as much as possible.
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| − | <div class="texttitle">Method</div>
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| − | <p>This year’s interlab study aims to reduce a major source of variability during fluorescence measurements: the number of cells in the sample. Normally, we take the Abs600 of a sample as a representation of its cell density, but still many previous studies showed that there was significant lab-to-lab variability(?). Therefore, we followed the protocols delivered by the iGEM authority and carried out this verification.</p>
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| − | <br>
| + | |
| − | <p>According to the protocol, we performed three calibration experiments (OD600 reference point, particle standard curve and the fluorescence standard curve) before the body part began. Results shown below. </p>
| + | |
| − | <br>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/0/06/T--BNU-China--image_interlab_1.jpg">
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| − | <p style="text-align:center;"><b>Table 1. OD600 reference point</b></p>
| + | |
| − | <br>
| + | |
| − | <p>4 replicates are measured respectively to acquire the average Abs600 of LUDOX CL-X solution and dH2O. Reference OD600 is given by the iGEM authority. The ratio of OD600/Abs600 is 3.652. </p>
| + | |
| − | <br>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/5/55/T--BNU-China--image_interlab_2.jpg">
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| − | <p style="text-align:center;"><b>Figure 1A Particle Standard Curve</b></p>
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| − | <img src="https://static.igem.org/mediawiki/2018/4/44/T--BNU-China--image_interlab_3.jpg">
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| − | <p style="text-align:center;"><b>Figure 1B Particle Standard Curve (log scale)</b></p>
| + | |
| − | <p>We use the silica beads delivered by iGEM and made a serial dilution in the 96-well plate according to the protocol. 4 replicates were measured in total. Origin data presented at the bottom of this page.
| + | |
| − | </p>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/b/b1/T--BNU-China--image_interlab_4.jpg">
| + | |
| − | <p style="text-align:center;"><b>Figure 2A Fluorescein Standard Curve</b></p>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2018/b/b1/T--BNU-China--image_interlab_5.jpg">
| + | |
| − | <p style="text-align:center;"><b>Figure 2B Fluorescein Standard Curve(log scale)</b></p>
| + | |
| − | <p>We use the fluorescein powder delivered by iGEM and made a serial dilution in the 96-well plate according to the protocol. 4 replicates were measured in total. Origin data presented at the bottom of this page.</p>
| + | |
| − | <p>After finishing the calibration, we carried out the cell culturing and the measurements of Abs600 and fluorescein. </p>
| + | |
| − | <p>First of all, we transformed the given plasmids into E.coli K-12 DH5-alpha on the plates of LB medium with chloramphenicol of 25μg/ml in 37℃, and picked 2 colonies of each plate over night, inoculating into 5ml Luria- Bertani liquid medium with 25 μg/mL chloramphenicol, incubating in 37°C at the speed of 220 rpm for 16hrs. </p>
| + | |
| − | <p>Secondly, the cell cultures were diluted to the concentration of Abs600=0.02 for target start and were transferred to the same LB medium of 12mL in 50mL falcon tubes, at the same incubator above. At the time of 0 and 6 hours of incubation, we took off 500μL into 1.5 ml eppendorf tubes of each colony of 8 devices, and placed them on ice to prevent from further growth.</p>
| + | |
| − | <p>Last but most importantly, we pipetted 4 replicates samples of each devices, 100μL per well, into a 96-well plate. Samples were laid out according to the plate diagram shown in the protocol.</p>
| + | |
| − |
| + | |
| − | <a id="sequencing"></a>
| + | |
| − | <br>
| + | |
| − | <div class="texttitle">Results</div>
| + | |
| − | <h3 class="classic-title";"><span>Sequencing</span></h3>
| + | |
| − | <p> • Device 1: J23101+I13504<br>
| + | |
| − | • Device 2: J23106+I13504<br>
| + | |
| − | • Device 3: J23117+I13504<br>
| + | |
| − | • Positive Control Device: I20270 in pSB1C3<br>
| + | |
| − | • Negative Control Device: R0040 in pSB1C3 </p>
| + | |
| − | <p>
| + | |
| − | >BBa_J23101 Part-only sequence (35 bp)<br>
| + | |
| − | <span style="padding-left:45px">Tttacagctagctcagtcctaggtattatgctagc</span></p><p>
| + | |
| − | >BBa_J23106 Part-only sequence (35 bp)<br>
| + | |
| − | <span style="padding-left:45px">Tttacggctagctcagtcctaggtatagtgctagc</span></p><p>
| + | |
| − | >BBa_J23117 Part-only sequence (35 bp)<br>
| + | |
| − | <span style="padding-left:45px">Ttgacagctagctcagtcctagggattgtgctagc</span></p><p>
| + | |
| − | >BBa_I13504 Part-only sequence (875 bp)<br>
| + | |
| − | <span style="padding-left:45px">Aaagaggagaaatactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata</span></p><p>
| + | |
| − | >BBa_I20270 Part-only sequence (919 bp)<br>
| + | |
| − | <span style="padding-left:45px">Ttgatggctagctcagtcctaggtacaatgctagctactagagtcacacaggaaagtactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata</span></p><p>
| + | |
| − | >BBa_R0040 Part-only sequence (54 bp)<br>
| + | |
| − | <span style="padding-left:45px">tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcac</span>
| + | |
| − | </p></p>
| + | |
| − |
| + | |
| − | <a id="data"></a>
| + | |
| − | <h3 class="classic-title";"><span>Data</span></h3>
| + | |
| − | <h4>OD600 Reference Point</h4>
| + | |
| − | <p><b>Table 1. OD600 Reference Point.</b></p>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2016/f/f4/T--Peking--image_interlab_3.png">
| + | |
| − | <br>
| + | |
| − | <h4>FITC Standard Curve</h4>
| + | |
| − | <p ><b>Table 2. Data of FITC standard curve.</b></p>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2016/1/1b/T--Peking--image_interlab_4.png">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2016/0/03/T--Peking--image_interlab_5.png">
| + | |
| − | <p ><b>Figure 2. FITC standard curve.</b></p>
| + | |
| − | <br>
| + | |
| − | <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">
| + | |
| − | <br>
| + | |
| − | <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">
| + | |
| − | <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/2016/7/71/T--Peking--image_interlab_9.png">
| + | |
| − | <p ><b>Figure 3. Blank subtraction and correction of Abs600 measurement.</b></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">
| + | |
| − | <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/2016/6/67/T--Peking--image_interlab_12.png">
| + | |
| − | <p ><b>Figure 4. Blank subtraction and correction of fluorescence measurement.</b></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">
| + | |
| − | <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/2016/0/07/T--Peking--image_interlab_15.png">
| + | |
| − | <p ><b>Figure 4. Average level of devices.</b></p>
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − |
| + | |
| − | | + | |
| − | <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>
| + | |
| − | | + | |
| − | <h3 class="classic-title";"><span>Appendix</span></h3>
| + | |
| − | <p>Individuals responsible for conducting InterLab study
| + | |
| − | • Dong Yiming measured the devices.
| + | |
| − | • Li Cheng processed the data.
| + | |
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