Difference between revisions of "Team:CCU Taiwan/Demonstrate"

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{{CCU_Taiwan}}
 
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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2017.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2017.igem.org/Judging/Awards"> award listed above</a>. </p>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2017.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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<body class="no-sidebar">
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<div id="page-wrapper">
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<!-- Header -->
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<div id="header">
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<!-- Inner -->
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<div class="inner">
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<header>
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<h1><a href="https://2017.igem.org/Team:CCU_Taiwan" id="logo">Demonstrate</a></h1>
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</header>
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</div>
 +
 
 +
<!-- Nav -->
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<nav id="nav">
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<ul>
 +
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 +
 
 +
<li>
 +
<a href="#">Project</a>
 +
<ul>
 +
<li><a href="#">Description</a></li>
 +
                                                                                <li>
 +
<a href="#">Biosensor</a>
 +
<ul>
 +
<li><a href="#">CSP detector</a></li>
 +
<li><a href="#">Lactate detector</a></li>
 +
</ul>
 +
</li>
 +
<li><a href="#">Modeling</a></li>
 +
<li><a href="#">Experiment</a></li>
 +
 +
<li><a href="Results">Result</a></li>
 +
<li><a href="#">Future perspective</a></li>
 +
<li><a href="#">Safty</a></li>
 +
</ul>
 +
</li>
 +
 
 +
<li>
 +
<a href="#">Dry lab</a>
 +
<ul>
 +
                                                                                <li>
 +
<a href="#">Hardware</a>
 +
<ul>
 +
<li><a href="#">Device</a></li>
 +
<li><a href="#">Detector</a></li>
 +
</ul>
 +
</li>
 +
 
 +
                                                                                <li>
 +
<a href="#">Software</a>
 +
<ul>
 +
<li><a href="#">IOT system</a></li>
 +
<li><a href="#">APP</a></li>
 +
                                                                                                <li><a href="#">Machine learning</a></li>
 +
</ul>
 +
</li>
 +
</ul>
 +
</li>
 +
 
 +
 
 +
<li>
 +
<a href="#">Human practice</a>
 +
<ul>
 +
<li><a href="#">Silver</a></li>
 +
<li><a href="#">Gold and Integrated</a></li>
 +
<li><a href="#">Engagemant</a></li>
 +
<li><a href="#">Entrepreneurship</a></li>
 +
</ul>
 +
</li>
 +
 
 +
<li>
 +
<a href="#">Part</a>
 +
<ul>
 +
<li><a href="#">Basic part</a></li>
 +
<li><a href="#">Composite part</a></li>
 +
 +
</ul>
 +
</li>
 +
 
 +
 
 +
<li>
 +
<a href="#">Notebook</a>
 +
<ul>
 +
<li><a href="#">Schedule</a></li>
 +
<li><a href="#">Protocol</a></li>
 +
<li><a href="https://2017.igem.org/Team:CCU_Taiwan/InterLab">InterLab</a></li>
 +
 +
</ul>
 +
</li>
 +
 
 +
 
 +
<li>
 +
<a href="#">Team</a>
 +
<ul>
 +
<li><a href="https://2017.igem.org/Team:CCU_Taiwan/Member">Member</a></li>
 +
<li><a href="https://2017.igem.org/Team:CCU_Taiwan/Collaborations">Collaborations</a></li>
 +
<li><a href="#">Attribution</a></li>
 +
 +
</ul>
 +
</li>
 +
 
 +
 
 +
<li>
 +
<a href="#">Judging</a>
 +
<ul>
 +
<li><a href="#">Gold</a></li>
 +
<li><a href="#">Silver</a></li>
 +
<li><a href="#">Bronze</a></li>
 +
 +
</ul>
 +
</li>
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<li><a href="no-sidebar.html">Q&A</a></li>
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</ul>
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</nav>
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</div>
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<!-- Main -->
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<div class="wrapper style1">
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<div class="container">
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<article id="main" class="special">
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<header>
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 +
<div class="div_nav">
 +
<nav class="toc_nav" id="toc_show">
 +
<ul>
 +
<li>
 +
<a href="#Fluorescein">Fluorescein curve</a>
 +
<ul>
 +
<li><a href="#Fluorescein-Plate-reader">Plate reader</a></li>
 +
<li><a href="#Fluorescein-Material">Material</a></li>
 +
<li><a href="#Fluorescein-Method">Method</a></li>
 +
<li><a href="#Fluorescein-Data-result">Data result</a></li>
 +
</ul>
 +
</li>
 +
<li>
 +
<a href="#OD600">OD600 Reference point</a>
 +
      <ul>
 +
<li><a href="#OD600-Plate-reader">Plate reader</a></li>
 +
<li><a href="#OD600-Material">Material</a></li>
 +
<li><a href="#OD600-Method">Method</a></li>
 +
<li><a href="#OD600-Data-result">Data result</a></li>
 +
      </ul>
 +
</li>
 +
 
 +
<li>
 +
<a href="#Cell">Cell measure</a>
 +
      <ul>
 +
<li><a href="#Cell-Material">Material</a></li>
 +
<li><a href="#Cell-Method">Method</a></li>
 +
<li><a href="#Cell-Data-result">Data result</a></li>
 +
      </ul>
 +
</li>
 +
 
 +
 
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</ul>
 +
  <svg class="toc-marker" width="200" height="200" xmlns="http://www.w3.org/2000/svg">
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    <path stroke="#444" stroke-width="3" fill="transparent" stroke-dasharray="0, 0, 0, 1000" stroke-linecap="round" stroke-linejoin="round" transform="translate(-0.5, -0.5)" />
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  </svg>
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</nav>
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<article class="contents_nav">
 +
  <section>
 +
 
 +
 
 +
<div id="Fluorescein">
 +
<h2>Fluorescein Fluorescence standard curve</h2>
 +
<div class="aaa"></div>
 
</div>
 
</div>
−
<div class="clear"></div>
 
  
 +
<div id="Fluorescein-Plate-reader">
  
−
<div class="column full_size">
+
<h3>Plate reader</h3>
−
<h1>Demonstrate</h1>
+
−
<h3>Gold Medal Criterion #4</h3>
+
  
 +
    <p>
 +
      microplate reader FLUOstar Omega</br>
 +
emission filter: 520 nm</br>
 +
excitation filter: 485 nm
 +
    </p>
 +
<div class="aaa"></div>
 +
</div>
 +
 
 +
<div id="Fluorescein-Material">
 +
 +
<h3>Material</h3>
 +
<p>
 +
Fluorescein sodium salt</br>
 +
1xPBS</br>
 +
Tissue culture testplate (black with flat bottom)
 +
</p>
 +
<div class="aaa"></div>
 +
</div>
 +
 +
<div id="Fluorescein-Method">
 +
 +
                <h3>Method</h3>
 +
 +
<ol><li>Prepare fluorescein stock solution</li></ol>
 
<p>
 
<p>
−
Teams that can show their system working under real world conditions are usually good at impressing the judges in iGEM. To achieve gold medal criterion #4, convince the judges that your project works. There are many ways in which your project working could be demonstrated, so there is more than one way to meet this requirement. This gold medal criterion was introduced in 2016, so check our what 2016 teams did to achieve a their gold medals!
+
1. Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.</br>
 +
2. Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS. </br>
 +
3. Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein  stock solution 50 μM </br></br>
 
</p>
 
</p>
−
 
+
<ol><li>Serial dilutions</li></ol>
 
<p>
 
<p>
−
Please see the <a href="https://2017.igem.org/Judging/Medals">2017 Medals Page</a> for more information.
+
1. Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12</br>
 +
2. Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1</br>
 +
3. Transfer 100 μl of fluorescein stock solution from A1 into A2. </br>
 +
4. Mix A2 by pipetting up and down 3x and transfer 100 μl into A3. </br>
 +
5. Mix A3 by pipetting up and down 3x and transfer 100 μl into A4. </br>
 +
6. Mix A4 by pipetting up and down 3x and transfer 100 μl into A5. </br>
 +
7. Mix A5 by pipetting up and down 3x and transfer 100 μl into A6. </br>
 +
8. Mix A6 by pipetting up and down 3x and transfer 100 μl into A7. </br>
 +
9. Mix A7 by pipetting up and down 3x and transfer 100 μl into A8. </br>
 +
10. Mix A8 by pipetting up and down 3x and transfer 100 μl into A9. </br>
 +
11. Mix A9 by pipetting up and down 3x and transfer 100 μl into A10.</br>
 +
12. Mix A10 by pipetting up and down 3x and transfer 100 μl into A11. </br>
 +
13. Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste.
 +
  (Caution: Do not to continue serial dilution into column 12.)</br>
 
</p>
 
</p>
 +
<ol><li>repeat serial dilute for Row B、D、E</strong></li></ol>
 +
<ol><li>Measure fluorescence of all samples in all standard measurement modes in instrument Record the data in your notebook</strong></li></ol>
 +
<ol><li>Import data into Excel (fluorescein standard curve tab ) Sheet_1 provided</li></ol>
 +
 +
<br/><br/>
 +
<div class="aaa"></div>
 +
</div>
 +
<div id="Fluorescein-Data-result">
  
 +
<h3>Data result</h3>
 +
<br/>
 +
<img src="https://static.igem.org/mediawiki/2017/8/85/FFs_1.png" style="display:block; margin:auto;"><br/><br/>
 +
<img src="https://static.igem.org/mediawiki/2017/a/a4/FFs_2.png" style="display:block; margin:auto;"><br/><br/>
 +
<img src="https://static.igem.org/mediawiki/2017/5/5c/FFs_3.png" style="display:block; margin:auto;"><br/>
 +
</div>
 +
</section>
  
 +
<section>
 +
 +
 +
<div id="OD600">
 +
<h2>OD600 Reference point</h2>
 +
<div class="aaa"></div>
 
</div>
 
</div>
  
  
−
<div class="column half_size">
+
<div id="OD600-Plate-reader">
  
−
<h4> What should we do for our demonstration?</h4>
+
<h3>Plate reader</h3>
  
−
<h5> Standard teams </h5>
+
    <p>
 +
Thermo Scientific™ Multiskan™ FC Filter-based Microplate Photometer</br>
 +
Filter: 595 nm</br>
 +
    </p>
 +
<div class="aaa"></div>
 +
</div>
 +
 
 +
<div id="OD600-Material">
  
−
<p>  
+
<h3>Material</h3>
−
If you have built a proof of concept system, you can demonstrate it working under real world conditions. If you have built a biological device that is intended to be a sensor, can you show it detecting whatever it is intended to sense. If it is intended to work in the field, you can show how this might work using a simulated version in the lab, or a simulation of your device in the field.<strong> Please note biological materials must not be taken out of the lab</strong>.
+
<p>
 +
1 ml LUDOX</br>
 +
mQH<sub>2</sub>O</br>
 +
96 well cell culture plate (clear with flat-bottom)
 +
</p>
 +
<div class="aaa"></div>
 +
</div>
 +
 
 +
<div id="OD600-Method">
 +
 
 +
                <h3>Method</h3>
 +
 
 +
<p>
 +
1. Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)</br>
 +
2. Add 100 μl of H<sub>2</sub>O into wells A2, B2, C2, D2 (or 1 mL H<sub>2</sub>O into cuvette)</br>
 +
3. Measure absorbance 600 nm of all samples in all standard measurement modes in instrument</br>
 +
4. Record the data in excel and Import data into Excel ( OD600 reference point tab ) Sheet_1 provided</br>
 
</p>
 
</p>
 +
<div class="aaa"></div>
 
</div>
 
</div>
  
−
<div class="column half_size">
+
<div id="OD600-Data-result">
  
−
<br>
+
<h3>Data result</h3>
−
<h5> Special track teams </h5>
+
<br/>
 +
<img src="https://static.igem.org/mediawiki/2017/5/5a/600_1.png" style="display:block; margin:auto;"><br/>
 +
</div>
 +
</section>
 +
 
 +
<section>
 +
 
 +
 
 +
<div id="Cell">
 +
<h2>Cell measure</h2>
 +
<div class="aaa"></div>
 +
</div>
 +
 
 +
 
 +
<div id="Cell-Material">
 +
 
 +
<h3>Material</h3>
 +
<p>
 +
Competent cells ( Escherichia coli strain DH5α)</br>
 +
LB (Luria Bertani) media</br>
 +
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 μg /mL)</br>
 +
50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)</br>
 +
Incubator at 37°C</br>
 +
1.5 ml eppendorf tubes for sample storage</br>
 +
Ice bucket with ice</br>
 +
Pipettes</br>
 +
96 well plate(cell culture 96 well plate、tissue culture testplate)</br>
 +
Devices (from InterLab Measurement Kit):</br>
 +
1. Negative control(BBa_R0040)</br>
 +
2. Positive control(J23151+B0032+E0040+B0010+B0012)</br>
 +
3. Test Device 1: J23101+I13504</br>
 +
4. Test Device 2: J23106+I13504</br>
 +
5. Test Device 3: J23117+I13504</br>
 +
6. Test Device 4: J23101+BCD2+E0040+B0015</br>
 +
7. Test Device 5: J23106+BCD2+E0040+B0015</br>
 +
8. Test Device 6: J23117+BCD2+E0040+B0015</br>
 +
</p>
 +
<div class="aaa"></div>
 +
</div>
 +
 
 +
<div id="Cell-Method">
 +
 
 +
                <h3>Method</h3>
  
 
<p>
 
<p>
−
Special track teams can achieve this medal criterion by bringing their work to the Jamboree and showcasing it in the track event. Art & Design, Measurement, Hardware and Software tracks will all have showcase events at the Giant Jamboree.<strong> Please note biological materials must not be taken out of the lab</strong>.
+
1. Day 1 : Resuspended each plasmid in plate 7 and transform into Escherichia coli DH5α.</br>
 +
&nbsp;(Transformation protocol is from iGEM)</br>
 +
2. Day 2 : Pick 2 colonies from each of plate and inoculate it on 5-10 mL LB medium +Chloramphenicol.Grow the cells overnight (16-18 hours) at 37°C and 170 rpm.</br>
 +
3. Day 3 : Set instrument to read OD600 (as OD calibration setting)and measure OD600 of the overnight cultures</br>
 +
4. Dilute the cultures to a target OD 600 of 0.02 in 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (covered with foil to block light).</br>
 +
5. Incubate the cultures at 37°C and 170 rpm.</br>
 +
6. Take 1000 μL samples of the cultures at 0, 2, 4, and 6 hours of incubation and place samples on ice</br>
 +
7. 4 replicates of 100 uL samples were taken from each culture at 0, 2, 4, and 6 hours of incubation and placed in a 96 well plate for OD and fluorescence measurements using the setup described above</br>
 
</p>
 
</p>
 +
<div class="aaa"></div>
 +
</div>
  
 +
<div id="Cell-Data-result">
  
 +
<h3>Data result</h3>
 +
<br/>
 +
<img src="https://static.igem.org/mediawiki/2017/3/30/Cell_1.png" style="display:block; margin:auto;"><br/><br/>
 +
<img src="https://static.igem.org/mediawiki/2017/f/fe/Cell_2.png" style="display:block; margin:auto;"><br/>
 
</div>
 
</div>
 +
</section>
 +
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</article>
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Revision as of 02:23, 27 October 2017

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Fluorescein Fluorescence standard curve

Plate reader

microplate reader FLUOstar Omega
emission filter: 520 nm
excitation filter: 485 nm

Material

Fluorescein sodium salt
1xPBS
Tissue culture testplate (black with flat bottom)

Method

  1. Prepare fluorescein stock solution

1. Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.
2. Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS.
3. Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 μM

  1. Serial dilutions

1. Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
2. Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1
3. Transfer 100 μl of fluorescein stock solution from A1 into A2.
4. Mix A2 by pipetting up and down 3x and transfer 100 μl into A3.
5. Mix A3 by pipetting up and down 3x and transfer 100 μl into A4.
6. Mix A4 by pipetting up and down 3x and transfer 100 μl into A5.
7. Mix A5 by pipetting up and down 3x and transfer 100 μl into A6.
8. Mix A6 by pipetting up and down 3x and transfer 100 μl into A7.
9. Mix A7 by pipetting up and down 3x and transfer 100 μl into A8.
10. Mix A8 by pipetting up and down 3x and transfer 100 μl into A9.
11. Mix A9 by pipetting up and down 3x and transfer 100 μl into A10.
12. Mix A10 by pipetting up and down 3x and transfer 100 μl into A11.
13. Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste. (Caution: Do not to continue serial dilution into column 12.)

  1. repeat serial dilute for Row B、D、E
  1. Measure fluorescence of all samples in all standard measurement modes in instrument Record the data in your notebook
  1. Import data into Excel (fluorescein standard curve tab ) Sheet_1 provided


Data result







OD600 Reference point

Plate reader

Thermo Scientific™ Multiskan™ FC Filter-based Microplate Photometer
Filter: 595 nm

Material

1 ml LUDOX
mQH2O
96 well cell culture plate (clear with flat-bottom)

Method

1. Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)
2. Add 100 μl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette)
3. Measure absorbance 600 nm of all samples in all standard measurement modes in instrument
4. Record the data in excel and Import data into Excel ( OD600 reference point tab ) Sheet_1 provided

Data result



Cell measure

Material

Competent cells ( Escherichia coli strain DH5α)
LB (Luria Bertani) media
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 μg /mL)
50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)
Incubator at 37°C
1.5 ml eppendorf tubes for sample storage
Ice bucket with ice
Pipettes
96 well plate(cell culture 96 well plate、tissue culture testplate)
Devices (from InterLab Measurement Kit):
1. Negative control(BBa_R0040)
2. Positive control(J23151+B0032+E0040+B0010+B0012)
3. Test Device 1: J23101+I13504
4. Test Device 2: J23106+I13504
5. Test Device 3: J23117+I13504
6. Test Device 4: J23101+BCD2+E0040+B0015
7. Test Device 5: J23106+BCD2+E0040+B0015
8. Test Device 6: J23117+BCD2+E0040+B0015

Method

1. Day 1 : Resuspended each plasmid in plate 7 and transform into Escherichia coli DH5α.
 (Transformation protocol is from iGEM)
2. Day 2 : Pick 2 colonies from each of plate and inoculate it on 5-10 mL LB medium +Chloramphenicol.Grow the cells overnight (16-18 hours) at 37°C and 170 rpm.
3. Day 3 : Set instrument to read OD600 (as OD calibration setting)and measure OD600 of the overnight cultures
4. Dilute the cultures to a target OD 600 of 0.02 in 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (covered with foil to block light).
5. Incubate the cultures at 37°C and 170 rpm.
6. Take 1000 μL samples of the cultures at 0, 2, 4, and 6 hours of incubation and place samples on ice
7. 4 replicates of 100 uL samples were taken from each culture at 0, 2, 4, and 6 hours of incubation and placed in a 96 well plate for OD and fluorescence measurements using the setup described above

Data result