Difference between revisions of "Team:SKLMT-China/InterLab"

 
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        <div class="banner-content">
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            <h2 class="title">InterLab</h2>
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            <p class="content">Standard measurement makes research easy</p>
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                    <li><a href="#"><i class="fa fa-home">&thinsp;&thinsp;Home</i></a></li>
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                                &thinsp;&thinsp;InterLab
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                        <ul class="dropdown-menu">
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                            <li><a href="#s1" class="scrolly">Introduction</a></li>
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                            <li><a href="#s2" class="scrolly">Results</a></li>
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                            <li><a href="#s3" class="scrolly">Discussion</a></li>
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                            <li><a href="#s4" class="scrolly">Conclusion</a></li>
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<h1>InterLab</h1>
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<h3>Bronze Medal Criterion #4</h3>
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<p><b>Standard Tracks:</b> Participate in the Interlab Measurement Study and/or obtain new, high quality experimental characterization data for an existing BioBrick Part or Device and enter this information on that part's Main Page in the Registry. The part that you are characterizing must NOT be from a 2018 part number range.
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    </section>
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For teams participating in the <a href="https://2018.igem.org/Measurement/InterLab">InterLab study</a>, all work must be shown on this page.
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</p>
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        <div class="container">
 +
        <div class="paragraph shadow">
 +
            <h2 class="title" id="s1"> Introduction</h2>
 +
            <p>Over the past four years, iGEM has advanced the frontiers of science with the biggest interlaboratory studies ever done in synthetic biology. The goal of the fifth iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements.</p>
 +
            <p>This study aim to verify that whether CFUs could take the pace of OD during the exploration about green fluorescence protein’s production and cell growth in <i>E. coli.</i> DH5α cells. If measurements that are taken in different labs are no more variable than measurements taken within the same lab, this study will improve the measurement tools available to both the iGEM community and the synthetic biology community as a whole. </p>
 +
            <p> It is a valuable experiment for the development of synthetic biology. So, we decide to participate in this meaningful study.</p>
 
</div>
 
</div>
 +
<div class="paragraph shadow">
 +
            <h2 class="title" id="s2"> Results</h2>
 +
            <h3 class="title">1. Transformation of 8 test devices into competent <i>E. coli.</i>DH5α.</h3>
 +
            <p>The transformation was successful for all the plasmids and resulted in a proper amount of colonies on all plates. After that, two colonies from each plate were picked and inoculated in LB medium containing chloramphenicol overnight (approximately 16-18 hours) at 37°C and 220 rpm. </p>
 +
            <p><b>Information about the devices:</b></p>
  
 +
            <div class="table-wrapper">
 +
                <table class="alt">
 +
                    <thead>
 +
                        <tr>
 +
                            <th>Devices</th>
 +
                            <th>Part Number </th>
 +
                        </tr>
 +
                    </thead>
 +
                    <tbody>
 +
                        <tr>
 +
                            <td>Positive Control</td>
 +
                            <td>BBa_I20270</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>Negative Control</td>
 +
                            <td>BBa_R0040</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>Test Device 1</td>
 +
                            <td>BBa_J364000</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>Test Device 2</td>
 +
                            <td>BBa_J364001</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>Test Device 3</td>
 +
                            <td>BBa_J364002</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>Test Device 4</td>
 +
                            <td>BBa_J364007</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>Test Device 5</td>
 +
                            <td>BBa_J364008</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>Test Device 6</td>
 +
                            <td>BBa_J364009</td>
 +
                        </tr>
 +
                    </tbody>         
 +
                </table>
 +
            </div>
  
 +
            <h3 class="title">2. Measurement of LUDOX CL-X OD600 Reference Point.</h3>
 +
            <p>LUDOX CL-X (45% colloidal silica suspension) was used as a single point reference to obtain a ratiometric conversion factor which allows us to  transform absorbance (Abs600) data into a comparable OD 600 measurement. </p>
 +
            <p>It’s necessary for this study. Because, generally, plate readers do not have a 1 cm path length which means that measurements are volume dependent. However, to be convictive, measurements must be transformed to be standard.</p>
 +
            <p> By doing so, we obtained following data.</p>
  
 +
            <div class="table-wrapper">
 +
                <table class="alt">
 +
                    <thead>
 +
                        <tr>
 +
                            <th>Measurement</th>
 +
                            <th>LUDOX CL-X </th>
 +
                            <th>H2O </th>
 +
                        </tr>
 +
                    </thead>
 +
                    <tbody>
 +
                        <tr>
 +
                            <td>Replicate 1</td>
 +
                            <td>0.057</td>
 +
                            <td>0.037 </td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>Replicate 2</td>
 +
                            <td>0.057</td>
 +
                            <td> 0.036</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>Replicate 3</td>
 +
                            <td>0.057</td>
 +
                            <td>0.036</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>Replicate 4</td>
 +
                            <td>0.058</td>
 +
                            <td>0.036 </td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>Arith. Mean</td>
 +
                            <td>0.057</td>
 +
                            <td>0.036 </td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>Corrected Abs600</td>
 +
                            <td>0.021</td>
 +
                            <td></td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>Reference OD600</td>
 +
                            <td>0.063</td>
 +
                            <td> </td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>OD600/Abs600</td>
 +
                            <td>3.000</td>
 +
                            <td> </td>
 +
                        </tr>
  
  
 +
                    </tbody>
  
 +
                </table>
 +
            </div>
  
 +
            <h3 class="title">3. Graphing a Particle Standard Curve.</h3>
 +
 +
            <p>With the Microsphere Stock Solution and the serial dilution of Microspheres prepared, particle standard curve was measured. </p>
 +
            <span class="image fit">
 +
                <img  class="lb" src="https://static.igem.org/mediawiki/2018/6/67/T--SKLMT-China--_psc.png" alt="Figure1.3-1_The Abs600 a dilution series of monodisperse silica microspheres. " data-src="https://static.igem.org/mediawiki/2018/6/67/T--SKLMT-China--_psc.png"/>
 +
            </span>
 +
            <span class="image fit">
 +
                <img class="lb" src="https://static.igem.org/mediawiki/2018/5/5c/T--SKLMT-China--psc_log.png" alt="Figure1.3-2_The Abs600 a dilution series of monodisperse silica microspheres on a logarithmic scale."data-src="https://static.igem.org/mediawiki/2018/5/5c/T--SKLMT-China--psc_log.png" />
 +
            </span>
 +
 +
            <h3 class="title">4. Graphing a Fluorescence Standard Curve</h3>
 +
 +
            <p>To compare fluorescence output of test devices between teams, a standard curve for fluorescence of Fluorescein was measured. This stand curve is used to correct our cell-based readings to an equivalent Fluorescein concentration. </p>
 +
 +
            <p>Following the instructions, we obtained following results. </p>
 +
            <span class="image fit">
 +
                <img class="lb" src="https://static.igem.org/mediawiki/2018/f/fc/T--SKLMT-China--fluorescein.png" alt="Figure1.4-1_A standard curve generated by measuring the flourescence of the small molecule fluorescein. "data-src=" https://static.igem.org/mediawiki/2018/f/fc/T--SKLMT-China--fluorescein.png"/>
 +
            </span>
 +
            <span class="image fit">
 +
                <img class="lb" src="https://static.igem.org/mediawiki/2018/3/30/T--SKLMT-China--_fluorescein_log.png" alt="Figure1.4-2_A standard curve generated by measuring the flourescence of the small molecule fluorescein on a logarithmic scale."data-src="https://static.igem.org/mediawiki/2018/3/30/T--SKLMT-China--_fluorescein_log.png" />
 +
            </span>
 +
 +
            <h3 class="title">5. Measurement of OD600 and Fluorescence of Transformed Cells</h3>
 +
            <p>With Abs600=0.02 at the begining, colonies were incubated 37 ℃, 220rpm. Then they were measured on plate reader at 0 hour and 6 hour.</p>
 +
            <span class="image fit">
 +
                <img class="lb" src="https://static.igem.org/mediawiki/2018/5/5c/T--SKLMT-China--OD600.png" alt="Figure1.5-1_Calculated means from replicates for the OD600 after each time point."data-src="https://static.igem.org/mediawiki/2018/5/5c/T--SKLMT-China--OD600.png"/>
 +
            </span>
 +
            <span class="image fit">
 +
                <img class="lb" src="https://static.igem.org/mediawiki/2018/3/34/T--SKLMT-China--FM.png" alt="Figure1.5-2_Calculated means from both replicates for the fluorescence after each time point."data-src="https://static.igem.org/mediawiki/2018/3/34/T--SKLMT-China--FM.png"  />
 +
            </span>
 +
 +
            <h3 class="title">6. Measurement of Colony Forming Units per 0.1 OD600 <i>E. coli </i>cultures</h3>
 +
            <p>Finished “Starting Sample Preparation”, we got the “Starting Sample” with a 0.1 OD600 measurement. Then following the instructions, we obtained a series of dilution, and we counted the CFUs of dilution 3, dilution4, and dilution5 of positive control and negative control on plates. </p>
 +
            <p>Colony Forming Units per 0.1 OD600 <i>E. coli </i> cultures: </p>
 +
 +
            <span class="image fit">
 +
                <img class="lb" src="https://static.igem.org/mediawiki/2018/5/59/T--SKLMT-China--CFU_positive.png" alt="Figure1.6-1_CFUs of positive control cultures."data-src="https://static.igem.org/mediawiki/2018/5/59/T--SKLMT-China--CFU_positive.png" />
 +
            </span>
 +
            <span class="image fit">
 +
                <img class="lb" src="https://static.igem.org/mediawiki/2018/c/cc/T--SKLMT-China--CFU_negative.png" alt="Figure1.6-2_CFUs of negative control cultures."data-src="https://static.igem.org/mediawiki/2018/c/cc/T--SKLMT-China--CFU_negative.png" />
 +
            </span>
 +
 +
            <span class="image fit">
 +
                <img class="lb" src="https://static.igem.org/mediawiki/2018/8/8a/T--SKLMT-China--_CFU.png" alt="Figure1.6-3_CFUs per 0.1 OD600 Positive Control (BBa_I20270) cultures and Negative Control (BBa_R0040) cultures." data-src="https://static.igem.org/mediawiki/2018/8/8a/T--SKLMT-China--_CFU.png"/>
 +
            </span>
 +
</div>
 +
<div class="paragraph shadow">
 +
 +
            <h2 class="title" id="s3"> Discussion </h2>
 +
            <p>In figure 1.5-2, the highest fluorescence date is measured from device 4, much stronger than positive control. Device 2 and positive control show a modest fluorescence intensity and device 5,6,1 show low fluorescence intensity, while test devices 3 have close fluorescence signal to negative control group.</p>
 +
          <p> Well, these devise have different data in fluorescence intensity, which helps us to learn about their characters. However, when CFUs of them are concerned about, convictive conclusion is not available in just one lab temporarily. Several factors, like different moisture capacity of plates, different methods in spreading plate, and different time spent in each simple ect. result in different CFUs dates. But, we consider that some conclusion meaningful would be drown with the help of the big data collected all participants. </p>
 +
<p></p>
 +
            <h2 class="title" id="s4">Conclusion </h2>
 +
            <p>According to measurements above, 8 test devices all works in <i>E. coli.</i> DH5α cells, but different characters were shown in this interlab study.The OD results tells us that cells with device 4 and cells with device 5 grow happier. As for the fluorescence intensity, device 4 is the strongest member and device 3 is weaker than others. </p>
 +
            <p>Inter lab study is successfully finished, which gives us a hand in our experiment design and means a lot for the development of synthetic biology. </p>
 +
        </div>
 +
</div>
 +
    </div>
 +
 +
 +
   
 
</html>
 
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{{SKLMT-China/footer}}

Latest revision as of 21:11, 17 October 2018

  1.   Home
  2.   InterLab

Introduction

Over the past four years, iGEM has advanced the frontiers of science with the biggest interlaboratory studies ever done in synthetic biology. The goal of the fifth iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements.

This study aim to verify that whether CFUs could take the pace of OD during the exploration about green fluorescence protein’s production and cell growth in E. coli. DH5α cells. If measurements that are taken in different labs are no more variable than measurements taken within the same lab, this study will improve the measurement tools available to both the iGEM community and the synthetic biology community as a whole.

It is a valuable experiment for the development of synthetic biology. So, we decide to participate in this meaningful study.

Results

1. Transformation of 8 test devices into competent E. coli.DH5α.

The transformation was successful for all the plasmids and resulted in a proper amount of colonies on all plates. After that, two colonies from each plate were picked and inoculated in LB medium containing chloramphenicol overnight (approximately 16-18 hours) at 37°C and 220 rpm.

Information about the devices:

Devices Part Number
Positive Control BBa_I20270
Negative Control BBa_R0040
Test Device 1 BBa_J364000
Test Device 2 BBa_J364001
Test Device 3 BBa_J364002
Test Device 4 BBa_J364007
Test Device 5 BBa_J364008
Test Device 6 BBa_J364009

2. Measurement of LUDOX CL-X OD600 Reference Point.

LUDOX CL-X (45% colloidal silica suspension) was used as a single point reference to obtain a ratiometric conversion factor which allows us to transform absorbance (Abs600) data into a comparable OD 600 measurement.

It’s necessary for this study. Because, generally, plate readers do not have a 1 cm path length which means that measurements are volume dependent. However, to be convictive, measurements must be transformed to be standard.

By doing so, we obtained following data.

Measurement LUDOX CL-X H2O
Replicate 1 0.057 0.037
Replicate 2 0.057 0.036
Replicate 3 0.057 0.036
Replicate 4 0.058 0.036
Arith. Mean 0.057 0.036
Corrected Abs600 0.021
Reference OD600 0.063
OD600/Abs600 3.000

3. Graphing a Particle Standard Curve.

With the Microsphere Stock Solution and the serial dilution of Microspheres prepared, particle standard curve was measured.

Figure1.3-1_The Abs600 a dilution series of monodisperse silica microspheres. Figure1.3-2_The Abs600 a dilution series of monodisperse silica microspheres on a logarithmic scale.

4. Graphing a Fluorescence Standard Curve

To compare fluorescence output of test devices between teams, a standard curve for fluorescence of Fluorescein was measured. This stand curve is used to correct our cell-based readings to an equivalent Fluorescein concentration.

Following the instructions, we obtained following results.

Figure1.4-1_A standard curve generated by measuring the flourescence of the small molecule fluorescein. Figure1.4-2_A standard curve generated by measuring the flourescence of the small molecule fluorescein on a logarithmic scale.

5. Measurement of OD600 and Fluorescence of Transformed Cells

With Abs600=0.02 at the begining, colonies were incubated 37 ℃, 220rpm. Then they were measured on plate reader at 0 hour and 6 hour.

Figure1.5-1_Calculated means from replicates for the OD600 after each time point. Figure1.5-2_Calculated means from both replicates for the fluorescence after each time point.

6. Measurement of Colony Forming Units per 0.1 OD600 E. coli cultures

Finished “Starting Sample Preparation”, we got the “Starting Sample” with a 0.1 OD600 measurement. Then following the instructions, we obtained a series of dilution, and we counted the CFUs of dilution 3, dilution4, and dilution5 of positive control and negative control on plates.

Colony Forming Units per 0.1 OD600 E. coli cultures:

Figure1.6-1_CFUs of positive control cultures. Figure1.6-2_CFUs of negative control cultures. Figure1.6-3_CFUs per 0.1 OD600 Positive Control (BBa_I20270) cultures and Negative Control (BBa_R0040) cultures.

Discussion

In figure 1.5-2, the highest fluorescence date is measured from device 4, much stronger than positive control. Device 2 and positive control show a modest fluorescence intensity and device 5,6,1 show low fluorescence intensity, while test devices 3 have close fluorescence signal to negative control group.

Well, these devise have different data in fluorescence intensity, which helps us to learn about their characters. However, when CFUs of them are concerned about, convictive conclusion is not available in just one lab temporarily. Several factors, like different moisture capacity of plates, different methods in spreading plate, and different time spent in each simple ect. result in different CFUs dates. But, we consider that some conclusion meaningful would be drown with the help of the big data collected all participants.

Conclusion

According to measurements above, 8 test devices all works in E. coli. DH5α cells, but different characters were shown in this interlab study.The OD results tells us that cells with device 4 and cells with device 5 grow happier. As for the fluorescence intensity, device 4 is the strongest member and device 3 is weaker than others.

Inter lab study is successfully finished, which gives us a hand in our experiment design and means a lot for the development of synthetic biology.

SKLMT

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