Difference between revisions of "Team:Shanghaitech/InterLab"
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| + | <li class="active"><a href="https://2017.igem.org/Team:Shanghaitech">Home</a></li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Project">Project</a> | ||
| + | <ul> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Overview">Overview</a></li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Background">Background</a></li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Experiment_and_Modeling">Experiment and Modeling</a></li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Device">Device</a></li> | ||
| + | </ul> | ||
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| + | <li><a href="#">Drylab</a> | ||
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| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Introduction">Introduction</a></li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/#">Model of parameter filling and simulation</a></li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/#">Theoretical Calculation of Modeling</a></li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/#">Model for Our Project</a></li> | ||
| + | </ul> | ||
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| + | <li><a href="#">HumanPractice</a> | ||
| + | <ul> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Overview">Overview</a></li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Public_Education">Public Education</a></li> | ||
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| + | </ul> | ||
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| + | <li><a href="#">Collaboration</a> | ||
| + | <ul> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Safety">Safety</a></li> | ||
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| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Meet-Up">Meet-Up</a></li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li><a href="#">Notebook</a> | ||
| + | <ul> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Calendar">Calendar</a></li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Protocol">Protocol</a></li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Parts">Parts</a></li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/RawMaterial">Raw Material</a></li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li><a href="#">Notebook</a> | ||
| + | <ul> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Calendar">Calendar</a></li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Pro">Protocol</a></li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Parts">Parts</a></li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/RawMaterial">Raw Material</a></li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li><a href="#">Team</a> | ||
| + | <ul> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/TeamMeber">Team Member</a></li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Attributions">Attribution</a></li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Acknowledgement">Ackknowledgement</a></li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Achievement">Achievement</a> | ||
| + | <ul> | ||
| + | <li><a href="https://2017.igem.org/Team:Shanghaitech/Achievement">Achievement</a></li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <div id="mySidenav" class="sidenav"> | ||
| + | <a href="javascript:void(0)" class="closebtn" onclick="closeNav()">×</a> | ||
| + | <a href="#">A</a> | ||
| + | <a href="#">S</a> | ||
| + | <a href="#">Cl</a> | ||
| + | <a href="#">Co</a> | ||
| + | </div> | ||
| + | |||
| + | <br> | ||
| + | <br> | ||
| + | <br> | ||
| + | <br> | ||
| + | <br> | ||
| + | <br> | ||
| + | <br> | ||
| + | |||
| + | <div class="content"> | ||
| + | <h4>Abstract</h4> | ||
| + | <br> | ||
| + | <p align="left" style="text-indent: 21pt;"> Repeatability is one of the most important part of biological experiment, however, it’s difficult to repeat the measurement data in different labs. As for the measurement of fluorescence, it can be influenced by various factors: bacterial strain, culture medium, plate reader and so on. Therefore, following a standard protocol strictly is one of the guarantee of the repeatable experimental conclusion. This year, we use plate reader rather than flow cytometry in 2016 (<a href="https://2016.igem.org/Team:ShanghaitechChina/InterLab">click here to view</a>). Except for individual settings, we follow the standard protocol by iGEM authority completely, the data is reliable.</br> | ||
| + | </p> | ||
| + | <p> | ||
| + | <h4><span>Plasmids and the prediction of results</span></h4> | ||
| + | <div align="center"> | ||
| + | <h4><font size="3">All the eight plasmids are shown below:</font></h4> | ||
| + | <br> | ||
| + | <img align="middle" src="https://static.igem.org/mediawiki/2017/d/d1/T--Shanghaitech--interlab1.jpg" width="800" height="400" alt=""></img> | ||
| + | </div> | ||
| + | <br> | ||
| + | <br> | ||
| + | <p align="left" style="text-indent: 21pt;">From the table above, there are three different constitutive promotors (pcons) and two different ribosome binding sites (RBSs), which form six different combinations as Test Device 1-8. </p> | ||
| + | <br> | ||
| + | <br> | ||
| + | <div align="center"> | ||
| + | <img align="middle" src="https://static.igem.org/mediawiki/2017/8/83/T--Shanghaitech--interlab2.jpg" width="250" height="400" alt=""> | ||
| + | </div> | ||
| + | <div align="center"> | ||
| + | <a href="http://parts.igem.org/Part:BBa_J23101">(http://parts.igem.org/Part:BBa_J23101)</a></img> | ||
| + | <br> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <br> | ||
| + | <br> | ||
| + | <div> | ||
| + | <p class="MsoNormal" align="left" style="text-indent: 21pt;"> | ||
| + | <span lang="EN-US">According | ||
to the data of iGEM parts, we know that the strength of these three pcons: J23101</span>＞<span lang="EN-US">J23106</span>＞ | to the data of iGEM parts, we know that the strength of these three pcons: J23101</span>＞<span lang="EN-US">J23106</span>＞ | ||
| − | + | <span lang="EN-US"> | |
J23117. However, there lacks the | J23117. However, there lacks the | ||
reliable data of the strength of pcon (J23151) and the comparison of three RBSs | reliable data of the strength of pcon (J23151) and the comparison of three RBSs | ||
| Line 197: | Line 336: | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal" align="left"> | |
| − | + | <span lang="EN-US"> <a name="OLE_LINK2"></a><a name="OLE_LINK1">Test Device 1 </a></span>＞ <span lang="EN-US">Test Device 2 </span>＞<span lang="EN-US"> Test Device 3</span> | |
| − | + | <span lang="EN-US"> | |
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal" align="left" style="text-indent: 21pt;"> | |
| − | + | <span lang="EN-US">Test Device 4 </span>＞ <span lang="EN-US">Test Device 5 </span>＞ | |
| − | + | <span lang="EN-US"> | |
Test Device 6 | Test Device 6 | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal" align="left" style="text-indent: 21pt;"> | |
| − | + | <span lang="EN-US"> | |
All the plasmid sequences (.dna file) can be download from: <a href="https://drive.google.com/file/d/0Bxb6U-RiSYsgQ1pTU0FsTG1SY1k/view?usp=sharing">https://drive.google.com/file/d/0Bxb6U-RiSYsgQ1pTU0FsTG1SY1k/view?usp=sharing</a> | All the plasmid sequences (.dna file) can be download from: <a href="https://drive.google.com/file/d/0Bxb6U-RiSYsgQ1pTU0FsTG1SY1k/view?usp=sharing">https://drive.google.com/file/d/0Bxb6U-RiSYsgQ1pTU0FsTG1SY1k/view?usp=sharing</a> | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | </div> | |
| − | + | <br> | |
| − | + | <br> | |
| − | + | <div class="col-xs-12"> | |
| − | + | <div class="dividerHeading"> | |
| − | + | <h4><span>Component cell strain & 96 well plate & Plate reader</span></h4> | |
| − | + | </div> | |
| − | + | <br> | |
| − | + | </div> | |
| − | + | <br> | |
| − | + | <p style="text-indent: 21pt;"> | |
| − | + | <strong>Component cell strain</strong> | |
| − | + | </p> | |
| − | + | <p style="text-indent: 37pt;"> | |
| − | + | Brand: 康为世纪 (cwbiotech) | |
| − | + | </p> | |
| − | + | <p style="text-indent: 37pt;"> | |
| − | + | Strain: <em>E.coli</em> K-12 DH5α | |
| − | + | </p> | |
| − | + | <p style="text-indent: 21pt;"> | |
| − | + | <strong>96 well plate</strong> | |
| − | + | </p> | |
| − | + | <p style="text-indent: 37pt;"> | |
| − | + | Brand: Corning | |
| − | + | </p> | |
| − | + | <p style="text-indent: 37pt;"> | |
| − | + | -Black plate | |
| − | + | </p> | |
| − | + | <p style="text-indent: 37pt;"> | |
| − | + | -Flat-bottomed wells | |
| − | + | </p> | |
| − | + | <p align="center"> | |
| − | + | <img width="265" height="354" src="https://static.igem.org/mediawiki/2017/thumb/9/97/T--Shanghaitech--interlab3.jpg/449px-T--Shanghaitech--interlab3.jpg" /> | |
| − | + | </p> | |
| − | + | <p align="center"> | |
| − | + | <img width="350" height="263" src="https://static.igem.org/mediawiki/2017/thumb/2/23/T--Shanghaitech--interlab5.jpg/320px-T--Shanghaitech--interlab5.jpg" /> | |
| − | + | </p> | |
| − | + | <p style="text-indent: 21pt;"> | |
| − | + | <strong>Plate reader</strong> | |
| − | + | </p> | |
| − | + | <p style="text-indent: 37pt;"> | |
| − | + | Brand: BioTek | |
| − | + | </p> | |
| − | + | <p style="text-indent: 37pt;"> | |
| − | + | Instrument model: Cytation 5 | |
| − | + | </p> | |
| − | + | <p style="text-indent: 37pt;"> | |
| − | + | Serial number: 1511021F | |
| − | + | </p> | |
| − | + | <p style="text-indent: 37pt;"> | |
| − | + | Pathlength correction: No | |
| − | + | </p> | |
| − | + | <p style="text-indent: 37pt;"> | |
| − | + | Number of flashes per well: 10 | |
| − | + | </p> | |
| − | + | <p style="text-indent: 37pt;"> | |
| − | + | Orbital averaging (mm): 3.5 | |
| − | + | </p> | |
| − | + | <p style="text-indent: 37pt;"> | |
| − | + | Fluorescence reading: top optic | |
| − | + | </p> | |
| − | + | <p style="text-indent: 37pt;"> | |
| − | + | Filter: Yes | |
| − | + | </p> | |
| − | + | <p style="text-indent: 37pt;"> | |
| − | + | Excitation wavelength (nm): 485/9 | |
| − | + | </p> | |
| − | + | <p style="text-indent: 37pt;"> | |
| − | + | Emission wavelength (nm): 528/9 | |
| − | + | </p> | |
| − | + | <p align="center"> | |
| − | + | <img width="426" height="567" src="https://static.igem.org/mediawiki/2017/thumb/8/86/T--Shanghaitech--interlab4.jpg/449px-T--Shanghaitech--interlab4.jpg" /> | |
| − | + | </p> | |
| − | + | <br> | |
| − | + | <br> | |
| − | + | <h2 align="center"> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
Protocol | Protocol | ||
</h2> | </h2> | ||
| − | + | <p class="MsoNormal"> | |
| − | + | <span lang="EN-US"> | |
(Some of the following steps are different | (Some of the following steps are different | ||
from standard protocol from iGEM authority, the changes and the reasons are | from standard protocol from iGEM authority, the changes and the reasons are | ||
shown as <span style="color:red">red</span>.) | shown as <span style="color:red">red</span>.) | ||
| − | + | <o:p></o:p> | |
| − | + | </span> | |
| − | + | </p> | |
| − | + | <p class="MsoNormal"><span lang="EN-US"> </span></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center"> | |
| − | + | <b> | |
<span lang="EN-US">Calibration </span>—— | <span lang="EN-US">Calibration </span>—— | ||
<span lang="EN-US"> | <span lang="EN-US"> | ||
| Line 323: | Line 452: | ||
</span> | </span> | ||
</b> | </b> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <b> | |
<span lang="EN-US"> | <span lang="EN-US"> | ||
Materials | Materials | ||
| Line 331: | Line 460: | ||
</span> | </span> | ||
</b> | </b> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <span lang="EN-US"> | |
1ml LUDOX | 1ml LUDOX | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <span lang="EN-US"> | |
ddH<sub>2</sub>O | ddH<sub>2</sub>O | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <span lang="EN-US"> | |
96 well plate | 96 well plate | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <b> | |
<span lang="EN-US"> | <span lang="EN-US"> | ||
Method | Method | ||
| Line 357: | Line 486: | ||
</span> | </span> | ||
</b> | </b> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Add 100 µl LUDOX into wells A1, B1, C1, D1 | Add 100 µl LUDOX into wells A1, B1, C1, D1 | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻<span lang="EN-US"> | |
Add 100 µl of H</span><sub><span style="font-family:"MS Gothic";mso-bidi-font-family: | Add 100 µl of H</span><sub><span style="font-family:"MS Gothic";mso-bidi-font-family: | ||
"MS Gothic""></span><span lang="EN-US">2</span></sub><span style="font-family: | "MS Gothic""></span><span lang="EN-US">2</span></sub><span style="font-family: | ||
"MS Gothic";mso-bidi-font-family:"MS Gothic""></span> | "MS Gothic";mso-bidi-font-family:"MS Gothic""></span> | ||
| − | + | <span lang="EN-US"> | |
O into | O into | ||
wells A2, B2, C2, D2 | wells A2, B2, C2, D2 | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Measure absorbance 600 nm of all samples in all standard measurement modes in instrument | Measure absorbance 600 nm of all samples in all standard measurement modes in instrument | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Record the data in the table below | Record the data in the table below | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻<span lang="EN-US"> | |
Import data into Excel (</span><span style="font-family:"MS Gothic";mso-bidi-font-family: | Import data into Excel (</span><span style="font-family:"MS Gothic";mso-bidi-font-family: | ||
"MS Gothic""></span><span lang="EN-US">OD600 reference point tab</span><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span> | "MS Gothic""></span><span lang="EN-US">OD600 reference point tab</span><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span> | ||
| − | + | <span lang="EN-US"> | |
) Sheet_1 provided | ) Sheet_1 provided | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"><span lang="EN-US"> </span></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center"> | |
| − | + | <b> | |
<span lang="EN-US">Calibration </span>—— | <span lang="EN-US">Calibration </span>—— | ||
<span lang="EN-US"> | <span lang="EN-US"> | ||
| Line 409: | Line 538: | ||
</span> | </span> | ||
</b> | </b> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <b> | |
<span lang="EN-US"> | <span lang="EN-US"> | ||
Materials | Materials | ||
| Line 417: | Line 546: | ||
</span> | </span> | ||
</b> | </b> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <span lang="EN-US"> | |
fluorescein | fluorescein | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <span lang="EN-US"> | |
10mL 1X PBS | 10mL 1X PBS | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <span lang="EN-US"> | |
96 well plate | 96 well plate | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <b> | |
<span lang="EN-US"> | <span lang="EN-US"> | ||
Method | Method | ||
| Line 443: | Line 572: | ||
</span> | </span> | ||
</b> | </b> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <b> | |
—— | —— | ||
<span lang="EN-US"> | <span lang="EN-US"> | ||
| Line 453: | Line 582: | ||
</span> | </span> | ||
</b> | </b> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Spin down fluorescein stock tube to make sure pellet is at the bottom of tube. | Spin down fluorescein stock tube to make sure pellet is at the bottom of tube. | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal" style="text-indent:15.75pt;mso-char-indent-count:1.5"> | |
| − | + | <span lang="EN-US" style="color:red"> | |
(5000rpm, 5min) | (5000rpm, 5min) | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Prepare 2X fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1X PBS. | Prepare 2X fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1X PBS. | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Dilute the 2X fluorescein stock solution with 1X PBS to make a 1X fluorescein solution and resulting concentration of fluorescein | Dilute the 2X fluorescein stock solution with 1X PBS to make a 1X fluorescein solution and resulting concentration of fluorescein | ||
stock solution 50 µM. | stock solution 50 µM. | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <b> | |
—— | —— | ||
<span lang="EN-US"> | <span lang="EN-US"> | ||
| Line 491: | Line 620: | ||
</span> | </span> | ||
</b> | </b> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻<span lang="EN-US"> | |
Add 100 µl of PBS</span><span style="font-family:"MS Gothic";mso-bidi-font-family: | Add 100 µl of PBS</span><span style="font-family:"MS Gothic";mso-bidi-font-family: | ||
"MS Gothic""></span> | "MS Gothic""></span> | ||
| − | + | <span lang="EN-US"> | |
into wells A2, B2, C2, D2....A12, B12, | into wells A2, B2, C2, D2....A12, B12, | ||
C12, D12 | C12, D12 | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻<span lang="EN-US"> | |
Add 200 µl</span><span style="font-family:"MS Gothic";mso-bidi-font-family: | Add 200 µl</span><span style="font-family:"MS Gothic";mso-bidi-font-family: | ||
"MS Gothic""></span> <span style="font-family:"MS Gothic";mso-bidi-font-family: | "MS Gothic""></span> <span style="font-family:"MS Gothic";mso-bidi-font-family: | ||
"MS Gothic""></span><span lang="EN-US">of fluorescein 1X stock</span><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span> | "MS Gothic""></span><span lang="EN-US">of fluorescein 1X stock</span><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span> | ||
| − | + | <span lang="EN-US"> | |
solution into A1, B1, C1, D1 | solution into A1, B1, C1, D1 | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Transfer 100 µl of fluorescein stock solution from A1 into A2. | Transfer 100 µl of fluorescein stock solution from A1 into A2. | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Mix A2 by pipetting up and down 3x and transfer 100 µl into A3… | Mix A2 by pipetting up and down 3x and transfer 100 µl into A3… | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Mix A3 by pipetting up and down 3x and transfer 100 µl into A4... | Mix A3 by pipetting up and down 3x and transfer 100 µl into A4... | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Mix A4 by pipetting up and down 3x and transfer 100 µl into A5... | Mix A4 by pipetting up and down 3x and transfer 100 µl into A5... | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Mix A5 by pipetting up and down 3x and transfer 100 µl into A6... | Mix A5 by pipetting up and down 3x and transfer 100 µl into A6... | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Mix A6 by pipetting up and down 3x and transfer 100 µl into A7... | Mix A6 by pipetting up and down 3x and transfer 100 µl into A7... | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Mix A7 by pipetting up and down 3x and transfer 100 µl into A8... | Mix A7 by pipetting up and down 3x and transfer 100 µl into A8... | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Mix A8 by pipetting up and down 3x and transfer 100 µl into A9... | Mix A8 by pipetting up and down 3x and transfer 100 µl into A9... | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Mix A9 by pipetting up and down 3x and transfer 100 µl into A10... | Mix A9 by pipetting up and down 3x and transfer 100 µl into A10... | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Mix A10 by pipetting up and down 3x and transfer 100 µl into A11... | Mix A10 by pipetting up and down 3x and transfer 100 µl into A11... | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻<span lang="EN-US"> | |
Mix A11 by pipetting up and down 3X and transfer 100 µl into </span><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span> | Mix A11 by pipetting up and down 3X and transfer 100 µl into </span><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span> | ||
| − | + | <span lang="EN-US"> | |
liquid waste | liquid waste | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Repeat dilution series for rows B, C, D | Repeat dilution series for rows B, C, D | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Measure fluorescence of all samples in all standard measurement modes in | Measure fluorescence of all samples in all standard measurement modes in | ||
instrument | instrument | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻ | |
| − | + | <span lang="EN-US"> | |
Record the data in your notebook | Record the data in your notebook | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | ◻<span lang="EN-US"> | |
Import data into Excel (fluorescein</span><span style="font-family:"MS Gothic"; | Import data into Excel (fluorescein</span><span style="font-family:"MS Gothic"; | ||
mso-bidi-font-family:"MS Gothic""></span><span lang="EN-US"> standard curve tab</span><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span> | mso-bidi-font-family:"MS Gothic""></span><span lang="EN-US"> standard curve tab</span><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span> | ||
| − | + | <span lang="EN-US"> | |
) Sheet_1 provided | ) Sheet_1 provided | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <br> | |
| − | + | <h2>Cell measurement</h2> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <b> | |
<span lang="EN-US"> | <span lang="EN-US"> | ||
Materials | Materials | ||
| Line 630: | Line 759: | ||
</span> | </span> | ||
</b> | </b> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <i><span lang="EN-US">E.coli</span></i><span lang="EN-US"> K-12 DH5</span>α | |
| − | + | <span lang="EN-US"> | |
component cells | component cells | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <span lang="EN-US"> | |
LB (Luria Bertani) medium | LB (Luria Bertani) medium | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <span lang="EN-US"> | |
Chloramphenicol (stock concentration<span style="color:red"> 30</span> mg/mL dissolved in EtOH - working stock <span style="color:red">30</span> ug/mL) | Chloramphenicol (stock concentration<span style="color:red"> 30</span> mg/mL dissolved in EtOH - working stock <span style="color:red">30</span> ug/mL) | ||
| − | + | <o:p></o:p> | |
| − | + | </span> | |
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <span lang="EN-US" style="color:red"> | |
(This concentration | (This concentration | ||
refers to one of our PIs, and it’s the common concentration in my lab.) | refers to one of our PIs, and it’s the common concentration in my lab.) | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <span lang="EN-US" style="color:red"> | |
(The file can be download | (The file can be download | ||
here: | here: | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <span lang="EN-US"> | |
<a href="https://drive.google.com/open?id=0Bxb6U-RiSYsgbjEzU0o4eVNYNWc">https://drive.google.com/open?id=0Bxb6U-RiSYsgbjEzU0o4eVNYNWc</a> | <a href="https://drive.google.com/open?id=0Bxb6U-RiSYsgbjEzU0o4eVNYNWc">https://drive.google.com/open?id=0Bxb6U-RiSYsgbjEzU0o4eVNYNWc</a> | ||
<span style="color:red"> | <span style="color:red"> | ||
| Line 671: | Line 800: | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </span> | |
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <span lang="EN-US" style="color:red"> | |
14 mL polypropylene | 14 mL polypropylene | ||
round-bottom tube with aluminum foil cover | round-bottom tube with aluminum foil cover | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <span lang="EN-US" style="color:red"> | |
(Comparing with 50mL | (Comparing with 50mL | ||
falcon tube, it is much more suitable for incubation, the reason is that its cover | falcon tube, it is much more suitable for incubation, the reason is that its cover | ||
| Line 687: | Line 816: | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <br> | |
| − | + | <br> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center"> | |
| + | <span lang="EN-US" style="color:red;mso-no-proof:yes"> | ||
<!--[if gte vml 1]> | <!--[if gte vml 1]> | ||
<v:shapetype id="_x0000_t75" | <v:shapetype id="_x0000_t75" | ||
| Line 725: | Line 855: | ||
</v:shape> | </v:shape> | ||
<![endif]--><!--[if !vml]--><img align="center" width="247" height="330" src="https://static.igem.org/mediawiki/2017/thumb/2/2e/T--Shanghaitech--interlab7.jpg/180px-T--Shanghaitech--interlab7.jpg" v:shapes="图片_x0020_8"><!--[endif]--><o:p></o:p></span></p> | <![endif]--><!--[if !vml]--><img align="center" width="247" height="330" src="https://static.igem.org/mediawiki/2017/thumb/2/2e/T--Shanghaitech--interlab7.jpg/180px-T--Shanghaitech--interlab7.jpg" v:shapes="图片_x0020_8"><!--[endif]--><o:p></o:p></span></p> | ||
| − | + | <br> | |
| − | + | <p class="MsoNormal"><span lang="EN-US">Incubator at 37</span>℃<span lang="EN-US"><o:p></o:p></span></p> | |
| − | + | <p class="MsoNormal"><span lang="EN-US">1.5 mL eppendorf tubes for sample storage<o:p></o:p></span></p> | |
| − | + | <p class="MsoNormal"><span lang="EN-US">Ice bucket with ice<o:p></o:p></span></p> | |
| − | + | <p class="MsoNormal"><span lang="EN-US">Pipettes<o:p></o:p></span></p> | |
| − | + | <p class="MsoNormal"><b><span lang="EN-US">Method<o:p></o:p></span></b></p> | |
| − | + | <p class="MsoNormal"><b><span lang="EN-US">Day | |
1: </span></b><span lang="EN-US">Transform </span><i><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span><span lang="EN-US">E.coli</span></i><i><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span></i><span lang="EN-US"> DH5</span>α<span lang="EN-US"> | 1: </span></b><span lang="EN-US">Transform </span><i><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span><span lang="EN-US">E.coli</span></i><i><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span></i><span lang="EN-US"> DH5</span>α<span lang="EN-US"> | ||
with eight kinds of plasmids from 2017 iGEM kit, plate 7.<o:p></o:p></span></p> | with eight kinds of plasmids from 2017 iGEM kit, plate 7.<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal"><b><span lang="EN-US">Day | |
2: </span></b><span lang="EN-US">Pick 2 colonies from each of plate and inoculate | 2: </span></b><span lang="EN-US">Pick 2 colonies from each of plate and inoculate | ||
it on 5 mL LB medium + chloramphenicol <span style="color:red">in 14mL | it on 5 mL LB medium + chloramphenicol <span style="color:red">in 14mL | ||
| − | polypropylene round-bottom tube.</span> Grow the cells overnight for 18 hours | + | polypropylene round-bottom tube.</span> Grow the cells overnight for 18 hours at 37°C and 220 rpm. |
| − | + | <o:p></o:p> | |
| − | + | </span> | |
| + | </p> | ||
| + | <p class="MsoNormal"><b><span lang="EN-US">Day | ||
3</span></b><b><span style="font-family: | 3</span></b><b><span style="font-family: | ||
"MS Gothic";mso-bidi-font-family:"MS Gothic""></span><span lang="EN-US">: </span></b><span lang="EN-US">Cell growth, sampling, and assay.<o:p></o:p></span></p> | "MS Gothic";mso-bidi-font-family:"MS Gothic""></span><span lang="EN-US">: </span></b><span lang="EN-US">Cell growth, sampling, and assay.<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal">◻<span lang="EN-US"> | |
Set the instrument to read OD600 (as OD calibration setting)<o:p></o:p></span></p> | Set the instrument to read OD600 (as OD calibration setting)<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal">◻<span lang="EN-US"> | |
Measure OD600 of the overnight cultures<o:p></o:p></span></p> | Measure OD600 of the overnight cultures<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal">◻<span lang="EN-US"> | |
Record data in the notebook<o:p></o:p></span></p> | Record data in the notebook<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal">◻<span lang="EN-US"> | |
Import data into Excel (</span><span style="font-family:"MS Gothic";mso-bidi-font-family: | Import data into Excel (</span><span style="font-family:"MS Gothic";mso-bidi-font-family: | ||
"MS Gothic""></span><span lang="EN-US">Dilution Calculation</span><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span><span lang="EN-US">) Sheet_1 provided<o:p></o:p></span></p> | "MS Gothic""></span><span lang="EN-US">Dilution Calculation</span><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span><span lang="EN-US">) Sheet_1 provided<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal">◻<span lang="EN-US"> | |
Dilute the cultures to a target OD</span><span style="font-family:"MS Gothic"; | Dilute the cultures to a target OD</span><span style="font-family:"MS Gothic"; | ||
mso-bidi-font-family:"MS Gothic""></span><span lang="EN-US">600 of 0.02 in </span><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span><span lang="EN-US" style="color:red">5 m</span><span style="font-family:"MS Gothic"; | mso-bidi-font-family:"MS Gothic""></span><span lang="EN-US">600 of 0.02 in </span><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span><span lang="EN-US" style="color:red">5 m</span><span style="font-family:"MS Gothic"; | ||
mso-bidi-font-family:"MS Gothic";color:red"></span><span lang="EN-US" style="color:red">L </span><span lang="EN-US">LB medium + Chloramphenicol 14mL | mso-bidi-font-family:"MS Gothic";color:red"></span><span lang="EN-US" style="color:red">L </span><span lang="EN-US">LB medium + Chloramphenicol 14mL | ||
polypropylene round-bottom tube with aluminum foil cover.<o:p></o:p></span></p> | polypropylene round-bottom tube with aluminum foil cover.<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal"><span lang="EN-US" style="color:red">(Overmuch cultures in | |
tube leads to anaerobic environment. Therefore, about 1/3 cultures here.)</span><span lang="EN-US"><o:p></o:p></span></p> | tube leads to anaerobic environment. Therefore, about 1/3 cultures here.)</span><span lang="EN-US"><o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal">◻<span lang="EN-US"> | |
Incubate the cultures at 37°C and 220 rpm.<o:p></o:p></span></p> | Incubate the cultures at 37°C and 220 rpm.<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal">◻<span lang="EN-US"> | |
Take 500 µL samples of the cultures at 0, 2, 4, and 6 hours of incubation.<o:p></o:p></span></p> | Take 500 µL samples of the cultures at 0, 2, 4, and 6 hours of incubation.<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal">◻<span lang="EN-US"> | |
Place samples on ice.<o:p></o:p></span></p> | Place samples on ice.<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal">◻<span lang="EN-US"> | |
At the end of sampling point, measure the samples (OD and Fl measurement).<o:p></o:p></span></p> | At the end of sampling point, measure the samples (OD and Fl measurement).<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal">◻<span lang="EN-US"> | |
Record data in the notebook.<o:p></o:p></span></p> | Record data in the notebook.<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal">◻<span lang="EN-US"> | |
Import data into Excel (</span><span style="font-family:"MS Gothic";mso-bidi-font-family: | Import data into Excel (</span><span style="font-family:"MS Gothic";mso-bidi-font-family: | ||
"MS Gothic""></span><span lang="EN-US">cell measurement tab</span><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span><span lang="EN-US">) Sheet_1 provided.<o:p></o:p></span></p> | "MS Gothic""></span><span lang="EN-US">cell measurement tab</span><span style="font-family:"MS Gothic";mso-bidi-font-family:"MS Gothic""></span><span lang="EN-US">) Sheet_1 provided.<o:p></o:p></span></p> | ||
| − | + | <h1 align="center">Results</h1> | |
| − | + | <h2 align="center">Calibration —— OD600 reference point</h2> | |
| − | + | <p class="MsoNormal"><span lang="EN-US"><!--[if gte vml 1]> | |
<v:shapetype | <v:shapetype | ||
id="_x0000_t75" coordsize="21600,21600" o:spt="75" o:preferrelative="t" | id="_x0000_t75" coordsize="21600,21600" o:spt="75" o:preferrelative="t" | ||
| Line 801: | Line 933: | ||
<![endif]--><!--[if !vml]--> | <![endif]--><!--[if !vml]--> | ||
<p align="center"><img align="center" width="599" height="180" src="https://static.igem.org/mediawiki/2017/a/a4/T--Shanghaitech--interlab8.jpg" v:shapes="图片_x0020_9"></p><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></p> | <p align="center"><img align="center" width="599" height="180" src="https://static.igem.org/mediawiki/2017/a/a4/T--Shanghaitech--interlab8.jpg" v:shapes="图片_x0020_9"></p><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal"><span lang="EN-US">Raw data link: <a href="https://drive.google.com/open?id=0Bxb6U-RiSYsgTFlyeVRGeXlscGs">https://drive.google.com/open?id=0Bxb6U-RiSYsgTFlyeVRGeXlscGs</a><o:p></o:p></span></p> | |
| − | + | <br> | |
| − | + | <br> | |
| − | + | <p class="MsoNormal"><span lang="EN-US"> </span></p> | |
| − | + | <h2 align="center">Calibration —— fluorescein fluorescence standard curve</h2> | |
| − | + | <br> | |
| + | <p class="MsoNormal"><span lang="EN-US"><!--[if gte vml 1]> | ||
<v:shape | <v:shape | ||
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| Line 813: | Line 946: | ||
</v:shape> | </v:shape> | ||
<![endif]--><!--[if !vml]--><p align="center"><img align="center" border="0" width="800" height="300" src="https://static.igem.org/mediawiki/2017/a/a0/T--Shanghaitech--interlab9.jpg" v:shapes="图片_x0020_10"></p><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></p> | <![endif]--><!--[if !vml]--><p align="center"><img align="center" border="0" width="800" height="300" src="https://static.igem.org/mediawiki/2017/a/a0/T--Shanghaitech--interlab9.jpg" v:shapes="图片_x0020_10"></p><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal"><span lang="EN-US">Raw data link: <a href="https://drive.google.com/open?id=0Bxb6U-RiSYsgVGl6RG1aQ1dxVFE">https://drive.google.com/open?id=0Bxb6U-RiSYsgVGl6RG1aQ1dxVFE</a><o:p></o:p></span></p> | |
| − | + | <br> | |
| − | + | <p class="MsoNormal"><span lang="EN-US"><!--[if gte vml 1]> | |
<v:shape | <v:shape | ||
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| Line 822: | Line 955: | ||
</v:shape> | </v:shape> | ||
<![endif]--><!--[if !vml]--><p align="center"><img border="0" width="554" height="424" src="https://static.igem.org/mediawiki/2017/thumb/f/f9/T--Shanghaitech--interlab10.jpg/314px-T--Shanghaitech--interlab10.jpg" v:shapes="图片_x0020_11"></p><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></p> | <![endif]--><!--[if !vml]--><p align="center"><img border="0" width="554" height="424" src="https://static.igem.org/mediawiki/2017/thumb/f/f9/T--Shanghaitech--interlab10.jpg/314px-T--Shanghaitech--interlab10.jpg" v:shapes="图片_x0020_11"></p><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></p> | ||
| − | + | <br> | |
| − | + | <p class="MsoNormal"><span lang="EN-US"> According | |
to the diagram above, we can notice that when the concentration of fluorescein | to the diagram above, we can notice that when the concentration of fluorescein | ||
is high (</span>＞<span lang="EN-US">10</span>μ<span lang="EN-US">M), the curve | is high (</span>＞<span lang="EN-US">10</span>μ<span lang="EN-US">M), the curve | ||
| Line 829: | Line 962: | ||
other iGEM team, as expected, this is a common question. Referring to some materials, | other iGEM team, as expected, this is a common question. Referring to some materials, | ||
we give two possible explanations:<o:p></o:p></span></p> | we give two possible explanations:<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal">——<span lang="EN-US">When concentration is too high, light cannot | |
pass through the sample to cause excitation, thus very high concentrations can | pass through the sample to cause excitation, thus very high concentrations can | ||
have very low fluorescence.<o:p></o:p></span></p> | have very low fluorescence.<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal">——<span lang="EN-US">The surface portion of sample nearest the | |
light absorbs too much light, little is available for the rest portion of the | light absorbs too much light, little is available for the rest portion of the | ||
sample; thus the readings will not be linear, though the measurement will be | sample; thus the readings will not be linear, though the measurement will be | ||
within the range of a calibration curve.<o:p></o:p></span></p> | within the range of a calibration curve.<o:p></o:p></span></p> | ||
| − | + | <br> | |
| − | + | <br> | |
| − | + | <h2 align="center">Cell measurement</h2> | |
| − | + | <br> | |
| + | <p class="MsoNormal"><span lang="EN-US">Raw data link: <a href="https://drive.google.com/open?id=0Bxb6U-RiSYsgbnNJRVpOaUJLZVU">https://drive.google.com/open?id=0Bxb6U-RiSYsgbnNJRVpOaUJLZVU</a><o:p></o:p></span></p> | ||
| + | <p class="MsoNormal" style="text-indent:21.0pt"><span lang="EN-US">All the raw data | ||
and following tables refer to the wells arrangement of iGEM protocal below:<o:p></o:p></span></p> | and following tables refer to the wells arrangement of iGEM protocal below:<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal" style="text-indent:21.0pt"><span lang="EN-US"> </span></p> | |
| − | + | <p class="MsoNormal" style="text-indent:21.0pt"><span lang="EN-US"> </span></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><span lang="EN-US"><!--[if gte vml 1]> | |
<v:shapetype id="_x0000_t75" | <v:shapetype id="_x0000_t75" | ||
coordsize="21600,21600" o:spt="75" o:preferrelative="t" path="m@4@5l@4@11@9@11@9@5xe" | coordsize="21600,21600" o:spt="75" o:preferrelative="t" path="m@4@5l@4@11@9@11@9@5xe" | ||
| Line 871: | Line 1,006: | ||
</v:shape> | </v:shape> | ||
<![endif]--><!--[if !vml]--><img width="476" height="386" src="https://static.igem.org/mediawiki/2017/b/ba/T--Shanghaitech--interlab11.jpg" v:shapes="图片_x0020_12"><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></p> | <![endif]--><!--[if !vml]--><img width="476" height="386" src="https://static.igem.org/mediawiki/2017/b/ba/T--Shanghaitech--interlab11.jpg" v:shapes="图片_x0020_12"><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal" style="text-indent:21.0pt"><span lang="EN-US"> </span></p> | |
| − | + | <h2><span lang="EN-US">Results:<o:p></o:p></span></h2> | |
| − | + | <p class="MsoNormal" style="text-indent:21.0pt"><span lang="EN-US"> </span></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><b><span lang="EN-US">OD600 </span>——<span lang="EN-US"> 0h<o:p></o:p></span></b></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><span lang="EN-US"><!--[if gte vml 1]> | |
<v:shape id="图片_x0020_13" | <v:shape id="图片_x0020_13" | ||
o:spid="_x0000_i1035" type="#_x0000_t75" style='width:415.8pt;height:86.4pt; | o:spid="_x0000_i1035" type="#_x0000_t75" style='width:415.8pt;height:86.4pt; | ||
| Line 883: | Line 1,018: | ||
</v:shape> | </v:shape> | ||
<![endif]--><!--[if !vml]--><img width="554" height="115" src="https://static.igem.org/mediawiki/2017/thumb/0/05/T--Shanghaitech--interlab12.jpg/799px-T--Shanghaitech--interlab12.jpg" v:shapes="图片_x0020_13"><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></p> | <![endif]--><!--[if !vml]--><img width="554" height="115" src="https://static.igem.org/mediawiki/2017/thumb/0/05/T--Shanghaitech--interlab12.jpg/799px-T--Shanghaitech--interlab12.jpg" v:shapes="图片_x0020_13"><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><b><span lang="EN-US">OD600 </span>——<span lang="EN-US"> 2h<o:p></o:p></span></b></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><span lang="EN-US"><!--[if gte vml 1]> | |
<v:shape id="图片_x0020_14" | <v:shape id="图片_x0020_14" | ||
o:spid="_x0000_i1034" type="#_x0000_t75" style='width:415.2pt;height:85.8pt; | o:spid="_x0000_i1034" type="#_x0000_t75" style='width:415.2pt;height:85.8pt; | ||
| Line 892: | Line 1,027: | ||
</v:shape> | </v:shape> | ||
<![endif]--><!--[if !vml]--><img width="554" height="114" src="https://static.igem.org/mediawiki/2017/thumb/9/9a/T--Shanghaitech--interlab13.jpg/800px-T--Shanghaitech--interlab13.jpg" v:shapes="图片_x0020_14"><!--[endif]--></span><b><span lang="EN-US"><o:p></o:p></span></b></p> | <![endif]--><!--[if !vml]--><img width="554" height="114" src="https://static.igem.org/mediawiki/2017/thumb/9/9a/T--Shanghaitech--interlab13.jpg/800px-T--Shanghaitech--interlab13.jpg" v:shapes="图片_x0020_14"><!--[endif]--></span><b><span lang="EN-US"><o:p></o:p></span></b></p> | ||
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><b><span lang="EN-US">OD600 </span>——<span lang="EN-US"> 4h<o:p></o:p></span></b></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><span lang="EN-US"><!--[if gte vml 1]> | |
<v:shape id="图片_x0020_15" | <v:shape id="图片_x0020_15" | ||
o:spid="_x0000_i1033" type="#_x0000_t75" style='width:415.2pt;height:87pt; | o:spid="_x0000_i1033" type="#_x0000_t75" style='width:415.2pt;height:87pt; | ||
| Line 901: | Line 1,036: | ||
</v:shape> | </v:shape> | ||
<![endif]--><!--[if !vml]--><img width="554" height="116" src="https://static.igem.org/mediawiki/2017/thumb/0/0c/T--Shanghaitech--interlab14.jpg/799px-T--Shanghaitech--interlab14.jpg" v:shapes="图片_x0020_15"><!--[endif]--></span><b><span lang="EN-US"><o:p></o:p></span></b></p> | <![endif]--><!--[if !vml]--><img width="554" height="116" src="https://static.igem.org/mediawiki/2017/thumb/0/0c/T--Shanghaitech--interlab14.jpg/799px-T--Shanghaitech--interlab14.jpg" v:shapes="图片_x0020_15"><!--[endif]--></span><b><span lang="EN-US"><o:p></o:p></span></b></p> | ||
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><b><span lang="EN-US">OD600 </span>——<span lang="EN-US"> 6h<o:p></o:p></span></b></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><span lang="EN-US"><!--[if gte vml 1]> | |
<v:shape id="图片_x0020_16" | <v:shape id="图片_x0020_16" | ||
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| Line 910: | Line 1,045: | ||
</v:shape> | </v:shape> | ||
<![endif]--><!--[if !vml]--><img width="554" height="115" src="https://static.igem.org/mediawiki/2017/thumb/7/77/T--Shanghaitech--interlab15.jpg/800px-T--Shanghaitech--interlab15.jpg" v:shapes="图片_x0020_16"><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></p> | <![endif]--><!--[if !vml]--><img width="554" height="115" src="https://static.igem.org/mediawiki/2017/thumb/7/77/T--Shanghaitech--interlab15.jpg/800px-T--Shanghaitech--interlab15.jpg" v:shapes="图片_x0020_16"><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal" style="text-indent:21.0pt"><span lang="EN-US"> </span></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><b><span lang="EN-US">Fluorescence </span>—— <span lang="EN-US">0h<o:p></o:p></span></b></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><span lang="EN-US"><!--[if gte vml 1]> | |
<v:shape id="图片_x0020_17" | <v:shape id="图片_x0020_17" | ||
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| Line 920: | Line 1,055: | ||
</v:shape> | </v:shape> | ||
<![endif]--><!--[if !vml]--><img width="554" height="115" src="https://static.igem.org/mediawiki/2017/thumb/0/0d/T--Shanghaitech--interlab16.jpg/800px-T--Shanghaitech--interlab16.jpg" v:shapes="图片_x0020_17"><!--[endif]--></span><b><span lang="EN-US"><o:p></o:p></span></b></p> | <![endif]--><!--[if !vml]--><img width="554" height="115" src="https://static.igem.org/mediawiki/2017/thumb/0/0d/T--Shanghaitech--interlab16.jpg/800px-T--Shanghaitech--interlab16.jpg" v:shapes="图片_x0020_17"><!--[endif]--></span><b><span lang="EN-US"><o:p></o:p></span></b></p> | ||
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><b><span lang="EN-US">Fluorescence </span>—— <span lang="EN-US">2h<o:p></o:p></span></b></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><span lang="EN-US"><!--[if gte vml 1]> | |
<v:shape id="图片_x0020_18" | <v:shape id="图片_x0020_18" | ||
o:spid="_x0000_i1030" type="#_x0000_t75" style='width:415.2pt;height:87pt; | o:spid="_x0000_i1030" type="#_x0000_t75" style='width:415.2pt;height:87pt; | ||
| Line 929: | Line 1,064: | ||
</v:shape> | </v:shape> | ||
<![endif]--><!--[if !vml]--><img width="554" height="116" src="https://static.igem.org/mediawiki/2017/thumb/b/bb/T--Shanghaitech--interlab17.jpg/800px-T--Shanghaitech--interlab17.jpg" v:shapes="图片_x0020_18"><!--[endif]--></span><b><span lang="EN-US"><o:p></o:p></span></b></p> | <![endif]--><!--[if !vml]--><img width="554" height="116" src="https://static.igem.org/mediawiki/2017/thumb/b/bb/T--Shanghaitech--interlab17.jpg/800px-T--Shanghaitech--interlab17.jpg" v:shapes="图片_x0020_18"><!--[endif]--></span><b><span lang="EN-US"><o:p></o:p></span></b></p> | ||
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><b><span lang="EN-US">Fluorescence </span>—— <span lang="EN-US">4h<o:p></o:p></span></b></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><span lang="EN-US"><!--[if gte vml 1]> | |
<v:shape id="图片_x0020_19" | <v:shape id="图片_x0020_19" | ||
o:spid="_x0000_i1029" type="#_x0000_t75" style='width:415.2pt;height:86.4pt; | o:spid="_x0000_i1029" type="#_x0000_t75" style='width:415.2pt;height:86.4pt; | ||
| Line 938: | Line 1,073: | ||
</v:shape> | </v:shape> | ||
<![endif]--><!--[if !vml]--><img width="554" height="115" src="https://static.igem.org/mediawiki/2017/thumb/0/0e/T--Shanghaitech--interlab18.jpg/800px-T--Shanghaitech--interlab18.jpg" v:shapes="图片_x0020_19"><!--[endif]--></span><b><span lang="EN-US"><o:p></o:p></span></b></p> | <![endif]--><!--[if !vml]--><img width="554" height="115" src="https://static.igem.org/mediawiki/2017/thumb/0/0e/T--Shanghaitech--interlab18.jpg/800px-T--Shanghaitech--interlab18.jpg" v:shapes="图片_x0020_19"><!--[endif]--></span><b><span lang="EN-US"><o:p></o:p></span></b></p> | ||
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><b><span lang="EN-US">Fluorescence </span>—— <span lang="EN-US">6h<o:p></o:p></span></b></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><span lang="EN-US"><!--[if gte vml 1]> | |
<v:shape id="图片_x0020_24" | <v:shape id="图片_x0020_24" | ||
o:spid="_x0000_i1028" type="#_x0000_t75" style='width:415.2pt;height:87pt; | o:spid="_x0000_i1028" type="#_x0000_t75" style='width:415.2pt;height:87pt; | ||
| Line 947: | Line 1,082: | ||
</v:shape> | </v:shape> | ||
<![endif]--><!--[if !vml]--><img width="554" height="116" src="https://static.igem.org/mediawiki/2017/thumb/c/cf/T--Shanghaitech--interlab19.jpg/800px-T--Shanghaitech--interlab19.jpg" v:shapes="图片_x0020_24"><!--[endif]--></span><b><span lang="EN-US"><o:p></o:p></span></b></p> | <![endif]--><!--[if !vml]--><img width="554" height="116" src="https://static.igem.org/mediawiki/2017/thumb/c/cf/T--Shanghaitech--interlab19.jpg/800px-T--Shanghaitech--interlab19.jpg" v:shapes="图片_x0020_24"><!--[endif]--></span><b><span lang="EN-US"><o:p></o:p></span></b></p> | ||
| − | + | <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US"> </span></b></p> | |
| − | + | <p class="MsoNormal" style="text-indent:21.0pt"><span lang="EN-US">The curve are | |
shown below:<o:p></o:p></span></p> | shown below:<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal" style="text-indent:21.0pt"><span lang="EN-US"> </span></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><b><span lang="EN-US">OD600 0</span>——<span lang="EN-US">6 h<o:p></o:p></span></b></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><span lang="EN-US"><!--[if gte vml 1]> | |
<v:shape id="图片_x0020_20" | <v:shape id="图片_x0020_20" | ||
o:spid="_x0000_i1027" type="#_x0000_t75" style='width:338.4pt;height:507.6pt; | o:spid="_x0000_i1027" type="#_x0000_t75" style='width:338.4pt;height:507.6pt; | ||
| Line 960: | Line 1,095: | ||
</v:shape> | </v:shape> | ||
<![endif]--><!--[if !vml]--><img width="451" height="677" src="https://static.igem.org/mediawiki/2017/thumb/2/2c/T--Shanghaitech--interlab20.jpg/399px-T--Shanghaitech--interlab20.jpg" v:shapes="图片_x0020_20"><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></p> | <![endif]--><!--[if !vml]--><img width="451" height="677" src="https://static.igem.org/mediawiki/2017/thumb/2/2c/T--Shanghaitech--interlab20.jpg/399px-T--Shanghaitech--interlab20.jpg" v:shapes="图片_x0020_20"><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal" style="text-indent:21.0pt"><span lang="EN-US">We find that | |
Positive Control and Test Device 1 have a lower OD600 value at 2h and 4h. | Positive Control and Test Device 1 have a lower OD600 value at 2h and 4h. | ||
According to the fluorescence curve, we suppose the more efficient GFP | According to the fluorescence curve, we suppose the more efficient GFP | ||
| Line 966: | Line 1,101: | ||
speaking, all the eight kinds of bacteria have the similar growth condition | speaking, all the eight kinds of bacteria have the similar growth condition | ||
during 0</span>——<span lang="EN-US">6 h.<o:p></o:p></span></p> | during 0</span>——<span lang="EN-US">6 h.<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><span lang="EN-US"> </span></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><b><span lang="EN-US">Fluorescence 0</span>——<span lang="EN-US">6 h<o:p></o:p></span></b></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><b><span lang="EN-US"><!--[if gte vml 1]> | |
<v:shape | <v:shape | ||
id="图片_x0020_21" o:spid="_x0000_i1026" type="#_x0000_t75" style='width:315pt; | id="图片_x0020_21" o:spid="_x0000_i1026" type="#_x0000_t75" style='width:315pt; | ||
| Line 976: | Line 1,111: | ||
</v:shape> | </v:shape> | ||
<![endif]--><!--[if !vml]--><img width="420" height="630" src="https://static.igem.org/mediawiki/2017/thumb/9/99/T--Shanghaitech--interlab21.jpg/399px-T--Shanghaitech--interlab21.jpg" v:shapes="图片_x0020_21"><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></b></p> | <![endif]--><!--[if !vml]--><img width="420" height="630" src="https://static.igem.org/mediawiki/2017/thumb/9/99/T--Shanghaitech--interlab21.jpg/399px-T--Shanghaitech--interlab21.jpg" v:shapes="图片_x0020_21"><!--[endif]--></span><span lang="EN-US"><o:p></o:p></span></b></p> | ||
| − | + | <p class="MsoNormal" style="text-indent:21.0pt"><span lang="EN-US">According to the | |
diagram above, we can find distinctly that Positive Control</span>、<span lang="EN-US"> Test Device 1 and Test Device 4 have more efficient GFP expression | diagram above, we can find distinctly that Positive Control</span>、<span lang="EN-US"> Test Device 1 and Test Device 4 have more efficient GFP expression | ||
than the others.<o:p></o:p></span></p> | than the others.<o:p></o:p></span></p> | ||
| − | + | <p class="MsoNormal" style="text-indent:21.0pt"><span lang="EN-US"> </span></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><b><span lang="EN-US">Fluorescence/OD600 0</span>——<span lang="EN-US">6 h<o:p></o:p></span></b></p> | |
| − | + | <p class="MsoNormal" align="center" style="text-align:center;text-indent:21.0pt"><span lang="EN-US"><!--[if gte vml 1]> | |
<v:shape id="图片_x0020_22" | <v:shape id="图片_x0020_22" | ||
o:spid="_x0000_i1025" type="#_x0000_t75" style='width:305.4pt;height:407.4pt; | o:spid="_x0000_i1025" type="#_x0000_t75" style='width:305.4pt;height:407.4pt; | ||
| Line 989: | Line 1,124: | ||
<![endif]--><!--[if !vml]--><img width="407" height="543" src="https://static.igem.org/mediawiki/2017/f/f9/T--Shanghaitech--interlab22.jpg" v:shapes="图片_x0020_22"><!--[endif]--> | <![endif]--><!--[if !vml]--><img width="407" height="543" src="https://static.igem.org/mediawiki/2017/f/f9/T--Shanghaitech--interlab22.jpg" v:shapes="图片_x0020_22"><!--[endif]--> | ||
</span> | </span> | ||
| − | + | <span lang="EN-US"> | |
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal" style="text-indent:21.0pt"> | |
| − | + | <span lang="EN-US"> | |
Generally speaking, | Generally speaking, | ||
the fluorescence/OD600 value means the amount of GFP in each bacterium, thus, | the fluorescence/OD600 value means the amount of GFP in each bacterium, thus, | ||
| Line 1,000: | Line 1,135: | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal"> | |
| − | + | <span lang="EN-US"> | |
the diagram shows the fluorescence/OD600 | the diagram shows the fluorescence/OD600 | ||
results among the six devices: | results among the six devices: | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal" align="left"> | |
| − | + | <span lang="EN-US"> Test Device 1 </span>＞ <span lang="EN-US">Test | |
Device 2 </span>＞ | Device 2 </span>＞ | ||
| − | + | <span lang="EN-US"> | |
Test Device 3 | Test Device 3 | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal" align="left" style="text-indent: 21pt;"> | |
| − | + | <span lang="EN-US">Test Device 4 </span>＞ <span lang="EN-US">Test Device 5 </span>＞ | |
| − | + | <span lang="EN-US"> | |
Test Device 6 | Test Device 6 | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
| − | + | </p> | |
| − | + | <p class="MsoNormal" style="text-indent:21.0pt"> | |
| − | + | <span lang="EN-US"> | |
The results correspond | The results correspond | ||
to the prediction, which means that our data is strict and reliable. | to the prediction, which means that our data is strict and reliable. | ||
<o:p></o:p> | <o:p></o:p> | ||
</span> | </span> | ||
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Revision as of 11:28, 26 October 2017
Abstract
Repeatability is one of the most important part of biological experiment, however, it’s difficult to repeat the measurement data in different labs. As for the measurement of fluorescence, it can be influenced by various factors: bacterial strain, culture medium, plate reader and so on. Therefore, following a standard protocol strictly is one of the guarantee of the repeatable experimental conclusion. This year, we use plate reader rather than flow cytometry in 2016 (click here to view). Except for individual settings, we follow the standard protocol by iGEM authority completely, the data is reliable.
Plasmids and the prediction of results
All the eight plasmids are shown below:
From the table above, there are three different constitutive promotors (pcons) and two different ribosome binding sites (RBSs), which form six different combinations as Test Device 1-8.
According
to the data of iGEM parts, we know that the strength of these three pcons: J23101＞J23106＞
J23117. However, there lacks the
reliable data of the strength of pcon (J23151) and the comparison of three RBSs
(B0032, B0034, J364100). In conclusion, we predict the result of the Interlab fluorescence
measurement:
Test Device 1 ＞ Test Device 2 ＞ Test Device 3
Test Device 4 ＞ Test Device 5 ＞
Test Device 6
All the plasmid sequences (.dna file) can be download from: https://drive.google.com/file/d/0Bxb6U-RiSYsgQ1pTU0FsTG1SY1k/view?usp=sharing
Component cell strain & 96 well plate & Plate reader
Component cell strain
Brand: 康为世纪 (cwbiotech)
Strain: E.coli K-12 DH5α
96 well plate
Brand: Corning
-Black plate
-Flat-bottomed wells
Plate reader
Brand: BioTek
Instrument model: Cytation 5
Serial number: 1511021F
Pathlength correction: No
Number of flashes per well: 10
Orbital averaging (mm): 3.5
Fluorescence reading: top optic
Filter: Yes
Excitation wavelength (nm): 485/9
Emission wavelength (nm): 528/9
Protocol
(Some of the following steps are different
from standard protocol from iGEM authority, the changes and the reasons are
shown as red.)
Calibration ——
OD600 reference
point
Materials
1ml LUDOX
ddH2O
96 well plate
Method
◻
Add 100 µl LUDOX into wells A1, B1, C1, D1
◻
Add 100 µl of H2
O into
wells A2, B2, C2, D2
◻
Measure absorbance 600 nm of all samples in all standard measurement modes in instrument
◻
Record the data in the table below
◻
Import data into Excel (OD600 reference point tab
) Sheet_1 provided
Calibration ——
fluorescein
fluorescence standard curve
Materials
fluorescein
10mL 1X PBS
96 well plate
Method
——
Prepare
the fluorescein stock solution
◻
Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.
(5000rpm, 5min)
◻
Prepare 2X fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1X PBS.
◻
Dilute the 2X fluorescein stock solution with 1X PBS to make a 1X fluorescein solution and resulting concentration of fluorescein
stock solution 50 µM.
——
Prepare
the serial dilutions of fluorescein
◻
Add 100 µl of PBS
into wells A2, B2, C2, D2....A12, B12,
C12, D12
◻
Add 200 µl of fluorescein 1X stock
solution into A1, B1, C1, D1
◻
Transfer 100 µl of fluorescein stock solution from A1 into A2.
◻
Mix A2 by pipetting up and down 3x and transfer 100 µl into A3…
◻
Mix A3 by pipetting up and down 3x and transfer 100 µl into A4...
◻
Mix A4 by pipetting up and down 3x and transfer 100 µl into A5...
◻
Mix A5 by pipetting up and down 3x and transfer 100 µl into A6...
◻
Mix A6 by pipetting up and down 3x and transfer 100 µl into A7...
◻
Mix A7 by pipetting up and down 3x and transfer 100 µl into A8...
◻
Mix A8 by pipetting up and down 3x and transfer 100 µl into A9...
◻
Mix A9 by pipetting up and down 3x and transfer 100 µl into A10...
◻
Mix A10 by pipetting up and down 3x and transfer 100 µl into A11...
◻
Mix A11 by pipetting up and down 3X and transfer 100 µl into
liquid waste
◻
Repeat dilution series for rows B, C, D
◻
Measure fluorescence of all samples in all standard measurement modes in
instrument
◻
Record the data in your notebook
◻
Import data into Excel (fluorescein standard curve tab
) Sheet_1 provided
Cell measurement
Materials
E.coli K-12 DH5α
component cells
LB (Luria Bertani) medium
Chloramphenicol (stock concentration 30 mg/mL dissolved in EtOH - working stock 30 ug/mL)
(This concentration
refers to one of our PIs, and it’s the common concentration in my lab.)
(The file can be download
here:
https://drive.google.com/open?id=0Bxb6U-RiSYsgbjEzU0o4eVNYNWc
)
14 mL polypropylene
round-bottom tube with aluminum foil cover
(Comparing with 50mL
falcon tube, it is much more suitable for incubation, the reason is that its cover
isn’t completely closed which can make the oxygen entry freely, so the E.coli can be incubated in an aerobic environment.)
Incubator at 37℃
1.5 mL eppendorf tubes for sample storage
Ice bucket with ice
Pipettes
Method
Day
1: Transform E.coli DH5α
with eight kinds of plasmids from 2017 iGEM kit, plate 7.
Day
2: Pick 2 colonies from each of plate and inoculate
it on 5 mL LB medium + chloramphenicol in 14mL
polypropylene round-bottom tube. Grow the cells overnight for 18 hours at 37°C and 220 rpm.
Day
3: Cell growth, sampling, and assay.
◻
Set the instrument to read OD600 (as OD calibration setting)
◻
Measure OD600 of the overnight cultures
◻
Record data in the notebook
◻
Import data into Excel (Dilution Calculation) Sheet_1 provided
◻
Dilute the cultures to a target OD600 of 0.02 in 5 mL LB medium + Chloramphenicol 14mL
polypropylene round-bottom tube with aluminum foil cover.
(Overmuch cultures in
tube leads to anaerobic environment. Therefore, about 1/3 cultures here.)
◻
Incubate the cultures at 37°C and 220 rpm.
◻
Take 500 µL samples of the cultures at 0, 2, 4, and 6 hours of incubation.
◻
Place samples on ice.
◻
At the end of sampling point, measure the samples (OD and Fl measurement).
◻
Record data in the notebook.
◻
Import data into Excel (cell measurement tab) Sheet_1 provided.
Results
Calibration —— OD600 reference point
Raw data link: https://drive.google.com/open?id=0Bxb6U-RiSYsgTFlyeVRGeXlscGs
Calibration —— fluorescein fluorescence standard curve
Raw data link: https://drive.google.com/open?id=0Bxb6U-RiSYsgVGl6RG1aQ1dxVFE
According
to the diagram above, we can notice that when the concentration of fluorescein
is high (＞10μM), the curve
doesn’t follow linear relation. As for this problem, we have communicated with
other iGEM team, as expected, this is a common question. Referring to some materials,
we give two possible explanations:
——When concentration is too high, light cannot
pass through the sample to cause excitation, thus very high concentrations can
have very low fluorescence.
——The surface portion of sample nearest the
light absorbs too much light, little is available for the rest portion of the
sample; thus the readings will not be linear, though the measurement will be
within the range of a calibration curve.
Cell measurement
Raw data link: https://drive.google.com/open?id=0Bxb6U-RiSYsgbnNJRVpOaUJLZVU
All the raw data
and following tables refer to the wells arrangement of iGEM protocal below:
Results:
OD600 —— 0h
OD600 —— 2h
OD600 —— 4h
OD600 —— 6h
Fluorescence —— 0h
Fluorescence —— 2h
Fluorescence —— 4h
Fluorescence —— 6h
The curve are
shown below:
OD600 0——6 h
We find that
Positive Control and Test Device 1 have a lower OD600 value at 2h and 4h.
According to the fluorescence curve, we suppose the more efficient GFP
expression (strong promotor and RBS) leads to the less OD600 value. Generally
speaking, all the eight kinds of bacteria have the similar growth condition
during 0——6 h.
Fluorescence 0——6 h
According to the
diagram above, we can find distinctly that Positive Control、 Test Device 1 and Test Device 4 have more efficient GFP expression
than the others.
Fluorescence/OD600 0——6 h
Generally speaking,
the fluorescence/OD600 value means the amount of GFP in each bacterium, thus,
this value can represent the GFP expression strength than fluorescence value.
the diagram shows the fluorescence/OD600
results among the six devices:
Test Device 1 ＞ Test
Device 2 ＞
Test Device 3
Test Device 4 ＞ Test Device 5 ＞
Test Device 6
The results correspond
to the prediction, which means that our data is strict and reliable.
