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| | <link rel="stylesheet" type="text/css" href="https://2018.igem.org/wiki/index.php?title=Template:SJTU-BioX-Shanghai/interlab_CSS&action=raw&ctype=text/css" /> | | <link rel="stylesheet" type="text/css" href="https://2018.igem.org/wiki/index.php?title=Template:SJTU-BioX-Shanghai/interlab_CSS&action=raw&ctype=text/css" /> |
| | <link rel="stylesheet" type="text/css" href="https://2018.igem.org/wiki/index.php?title=Template:SJTU-BioX-Shanghai/footnote_CSS&action=raw&ctype=text/css" /> | | <link rel="stylesheet" type="text/css" href="https://2018.igem.org/wiki/index.php?title=Template:SJTU-BioX-Shanghai/footnote_CSS&action=raw&ctype=text/css" /> |
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| | <div class="content_nav_logo"> | | <div class="content_nav_logo"> |
| | <a href="" title="return to homepage"><img src="https://static.igem.org/mediawiki/2018/1/17/T--SJTU-BioX-Shanghai--content_nav_logo.png | | <a href="" title="return to homepage"><img src="https://static.igem.org/mediawiki/2018/1/17/T--SJTU-BioX-Shanghai--content_nav_logo.png |
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| − | "/> | + | <a title="Go to story page" href="https://2018.igem.org/Team:SJTU-BioX-Shanghai/Story"><img class="content_nav_b_story_badge" src="https://static.igem.org/mediawiki/2018/d/d0/T--SJTU-BioX-Shanghai--story_badge.png"></a> |
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| − | <p>We took part in the Fifth International Interlab Measurement Study which aims to determine if we can reduce lab-to-lab variability in fluorescence measurements by normalizing $\sum_{i=0}^N\int_{a}^{b}g(t,i)\text{d}t$ to absolute cell count or colony-forming units instead of | + | <p>We took part in the Fifth International Interlab Measurement Study that aims to clarify the possibility of reducing lab-to-lab variability in fluorescence measurements through normalizing to absolute cell count or colony-forming units instead of |
| | <span class="footnote_link">OD | | <span class="footnote_link">OD |
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| | Microorganism: Escherichia coli DH5α strains | | Microorganism: Escherichia coli DH5α strains |
| | </p> | | </p> |
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| | </a> | | </a> |
| | </h2> | | </h2> |
| − | <p>In order to compare data from different labs, all the teams were asked to follow the protocol provided by iGEM HQ. These can be found at:</p> | + | <p>In order to compare data from different labs, all teams were asked to follow the protocol provided by iGEM HQ, and can be found at:</p> |
| | <a title="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf" href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf">2018 Interlab Plate Reader Protocol</a> <br/> | | <a title="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf" href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf">2018 Interlab Plate Reader Protocol</a> <br/> |
| | <a title="http://parts.igem.org/Help:Protocols/Transformation" href="http://parts.igem.org/Help:Protocols/Transformation">Protocols/Transformation</a> | | <a title="http://parts.igem.org/Help:Protocols/Transformation" href="http://parts.igem.org/Help:Protocols/Transformation">Protocols/Transformation</a> |
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| | </a> | | </a> |
| | </h2> | | </h2> |
| − | <p>Before we took the cell measurements, we made three sets of unit calibration measurements.</p> | + | <p>Before performing cell measurements, we ruled out three sets of unit calibration measurements.</p> |
| − | <p>First, we used LUDOX CL-X as a single point reference to obtain a conversion factor to transform Abs600 data into a comparable OD<sub>600</sub> measurement. The conversion factor turns to be 3.111. </p> | + | <p>First, we used LUDOX CL-X as a single point reference to obtain a conversion factor to transform Abs<sub>600</sub> data into a comparable OD<sub>600</sub> measurement. The conversion factor turns to be 3.111. </p> |
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| − | <p>Then, we used a dilution series of monodisperse silica microspheres provided in kit and measured the Abs<sub>600</sub> of them to construct a standard curve of a particle concentration, which allows us to convert Abs<sub>600</sub> to an estimated number of cells.</p> | + | <p>Then, we’ve constructed a dilution series of monodisperse silica microspheres provided in kit and measured the Abs<sub>600</sub> of them. The results were used to construct a standard curve of a particle concentration that allows us to convert Abs<sub>600</sub> to an estimated number of cells.</p> |
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| − | <p>Last, we prepared a dilution series of fluorescein provided in kit and measure the fluorescence in our plate reader. By measuring these, we generated a standard curve of fluorescence for fluorescein concentration, which we used to convert the data we measured to equivalent fluorescein concen</p> | + | <p>At last, we’ve prepared a dilution series of fluorescein provided in kit and measure the fluorescence in our plate reader. By analyzing the data, we generated a standard curve of fluorescence for fluorescein concentration, enabling us to convert the data we measured to equivalent fluorescein concentration.</p> |
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| − | <p>In cell measurements, we measured the fluorescence and Abs<sub>600</sub> of all devices and blank samples at hour 0 and hour 6. The results are shown below:</p> | + | <p>In cell measurements, we measured the fluorescence and Abs<sub>600</sub> of all devices including blank samples at hour 0 and hour 6. The results are shown below:</p> |
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| | <div class="table_in_text"> | | <div class="table_in_text"> |
| | <p class="table_illustration">Table 1. Colony forming units per 0.1 OD<sub>600</sub></p> | | <p class="table_illustration">Table 1. Colony forming units per 0.1 OD<sub>600</sub></p> |
| − | <table style="border-collapse: collapse; "> | + | <table style="border-collapse: collapse;background-color: rgba(241,240,225, 0.7); "> |
| | <tr style="border-top:2px solid #000;"> | | <tr style="border-top:2px solid #000;"> |
| | <th rowspan="2">samples</th> | | <th rowspan="2">samples</th> |
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| | </a> | | </a> |
| | </h2> | | </h2> |
| − | <p> According to our data, device 4 showed the best fluorescence results, much better than the positive control. Device 1 was the second one with the highest emission. Device 3 showed lowest fluorescence emission, even lower than the negative control. Compared to the strength of all 6 devices’ promoter provided on the http://parts.igem.org/Part:BBa_J23101, we found that device 5 showed a rather low emission which was not consistent with the efficiency of its promoter. Since there were some problems with the transformation of device 5 from the very beginning, so probably the low fluorescence emission has something to do with the plasmid sequence. </p> | + | <p> According to our data, device 4 has shown the best fluorescence results which are even better than positive control while Device 1 was the second highest. On the contrary, Device 3 showed lowest fluorescence emission and even lower than negative control. Comparing with the strength of all 6 devices’ promoter provided on the <a title="" href="http://parts.igem.org/Part:BBa_J23101">Part: BBa_J23101</a>, we found that device 5 showed a rather low emission which was not consistent with the efficiency of its promoter. Since there were some problems with the transformation of device 5 from the very beginning, so it is possible that the low fluorescence emission has something to do with the plasmid sequence. </p> |
| − | <p>As for the conversion factor from OD to CFU, it is 9.51×108 CFU/mL in samples whose OD600 is 1 and Abs600 is 0.321 according to the conversion factor between OD600 and Abs600 while from the particle standard curve we obtained from the 2nd calibration experiment, the numbers of particles in samples whose Abs600 is 0.321 should be around 1.43×108. So there is still some difference between CFU and absorbance of cells in terms of computing the number of cells. | + | <p>As for the conversion factor from OD to CFU, we reach to the ratio of 9.51×10<sup>8</sup> CFU/mL in samples with OD600 = 1. And according to the conversion factor we calculated between OD<sub>600</sub> and Abs600, when OD<sub>600</sub> = 1, the corresponding Abs<sub>600</sub>=0.321. And based on the particle standard curve we obtained from the 2nd calibration experiment, the numbers of particles in samples with Abs<sub>600</sub> = 0.321 should be around 1.43×10<sup>8</sup>. So there is still difference between CFU and absorbance of cells in terms of computing the number of cells. </p> |
| − | </p> | + | |
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| | <div style="display: flex;flex-direction: row;justify-content: center;align-items: center;width:100%;height: 100%;"> | | <div style="display: flex;flex-direction: row;justify-content: center;align-items: center;width:100%;height: 100%;"> |
| − | <div style="width: auto;height: auto;"> | + | |
| | <div class="zoom_out_album"> | | <div class="zoom_out_album"> |
| | <img id="zoom_out_img"/> | | <img id="zoom_out_img"/> |
| | <p class="fig_illustration"></p> | | <p class="fig_illustration"></p> |
| − | <img class="close_icon"/> | + | |
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