Difference between revisions of "Team:SDU CHINA/interlab"

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<h3>Background</h3>
 
<h3>Background</h3>
 
<p>“All of the 2017 iGEM teams are invited and encouraged to participate in the Fourth International InterLaboratory Measurement Study in synthetic biology.” Our team took part in this study which aimed to be one of the labs to repeat measurements in different condition. In our case, we measured fluorescence using plate reader.</p>
 
<p>“All of the 2017 iGEM teams are invited and encouraged to participate in the Fourth International InterLaboratory Measurement Study in synthetic biology.” Our team took part in this study which aimed to be one of the labs to repeat measurements in different condition. In our case, we measured fluorescence using plate reader.</p>
 
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<p>This year's iGEM Interlab study is to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable and compare the results from various teams. iGEM Interlab study has provided 2 protocols of measuring GFP, and we choose “plate reader”. We have to measure absorbance at 600 nm and GFP fluorescence for GFP expression per cell for all devices.</p>
 
<p>This year's iGEM Interlab study is to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable and compare the results from various teams. iGEM Interlab study has provided 2 protocols of measuring GFP, and we choose “plate reader”. We have to measure absorbance at 600 nm and GFP fluorescence for GFP expression per cell for all devices.</p>
 
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<li>Test Device 6: J23117.BCD2.E0040.B0015 in pSB1C3 (Located in Kit Plate 6, well 20P)</li>
 
<li>Test Device 6: J23117.BCD2.E0040.B0015 in pSB1C3 (Located in Kit Plate 6, well 20P)</li>
 
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<h5>Strain used</h5>
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<ul>
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<li>Escherichia coli​ strain DH5α</li>
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<li>Materials</li>
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<li>1ml LUDOX</li>
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<li>H​2​0 </li>
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<li>96 well plate</li>
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<li>fluorescein </li>
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<li>10ml 1xPBS </li>
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<li>LB (Luria Bertani) media </li>
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<li>Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 ug/mL) </li>
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<li>50 ml Falcon tube </li>
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<li>Incubator at 37°C </li>
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<li>1.5 ml eppendorf tubes </li>
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<li>Ice bucket with ice </li>
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<li>Pipettes </li>
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<br>
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<h5>Machines</h5>
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<li>Thermo Multiskan FC</li>
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<li>BERTHOLD TECHNOLOGIES LB940Mith</li>
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<h3>Protocol</h3>
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<li><a href="#">Calibration Protocols</a></li>
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<li><a href="#">Cell measurement protocol</a></li>
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<h3>Results</h3>
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<h5>Sequencing</h5>
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<li>Positive Control ：BBa_I20270</li>
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<li>Negative Control ：BBa_R0040</li>
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<li>Test Device 1 ：BBa_J364000</li>
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<li>Test Device 2 ：BBa_J364001</li>
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<li>Test Device 3 ：BBa_J364002</li>
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<li>Test Device 4 ：BBa_J364003</li>
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<li>Test Device 5 ：BBa_J364004</li>
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<li>Test Device 6 ：BBa_J364005</li>
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<p><a href="https://2017.igem.org/Team:SDU_CHINA/interlab/data">>BBa_I20270 Part-only sequence (919 bp)</a></p>
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<br><br><br>
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<h3>Data</h3>
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<h5>OD600 Reference Point</h5>
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<p>Table 1. OD600 Reference Point.</p>
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<img src="">tabel
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<h5>Fluorescein Standard Curve</h5>
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<p>Table 2. Data of Fluorescein Standard Curve.</p>
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<img src="">tabel
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<h5>Normalization</h5>
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<p>Table 1. OD600 Reference Point.Table 3. Normalization.</p>
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<img src="">tabel5
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<h5>Cell Measurement</h5>
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<p>Table 4. Raw data of OD600 measurement.</p>
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<img src="">tabel
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Revision as of 01:11, 25 September 2017






Background

“All of the 2017 iGEM teams are invited and encouraged to participate in the Fourth International InterLaboratory Measurement Study in synthetic biology.” Our team took part in this study which aimed to be one of the labs to repeat measurements in different condition. In our case, we measured fluorescence using plate reader.




Design

We often used fluorescence as an indicator of promoter activity. However, the ability to repeat measurements in different labs has been difficult. To solve this problem, InterLab study has been developed as a robust measurement procedure for green fluorescent protein (GFP) which is one of the most used markers in synthetic biology.

This year's iGEM Interlab study is to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable and compare the results from various teams. iGEM Interlab study has provided 2 protocols of measuring GFP, and we choose “plate reader”. We have to measure absorbance at 600 nm and GFP fluorescence for GFP expression per cell for all devices.




Materials

Plasmids used
  • Positive Control Device: I20270 in pSB1C3 (Located in Kit Plate 6, well 20B)
  • Negative Control Device: R0040 in pSB1C3 (Located in Kit Plate 6, well 20D)
  • Test Device 1: J23101+I13504 in pSB1C3 (Located in Kit Plate 6, well 20F)
  • Test Device 2: J23106+I13504 in pSB1C3 (Located in Kit Plate 6, well 20H)
  • Test Device 3: J23117+I13504 in pSB1C3 (Located in Kit Plate 6, well 20J)
  • Test Device 4: J23101.BCD2.E0040.B0015 in pSB1C3 (Located in Kit Plate 6, well 20L)
  • Test Device 5: J23106.BCD2.E0040.B0015 in pSB1C3 (Located in Kit Plate 6, well 20N)
  • Test Device 6: J23117.BCD2.E0040.B0015 in pSB1C3 (Located in Kit Plate 6, well 20P)

Strain used
  • Escherichia coli​ strain DH5α
  • Materials
  • 1ml LUDOX
  • H​2​0
  • 96 well plate
  • fluorescein
  • 10ml 1xPBS
  • LB (Luria Bertani) media
  • Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH - working stock 25 ug/mL)
  • 50 ml Falcon tube
  • Incubator at 37°C
  • 1.5 ml eppendorf tubes
  • Ice bucket with ice
  • Pipettes

Machines
  • Thermo Multiskan FC
  • BERTHOLD TECHNOLOGIES LB940Mith



Protocol




Results

Sequencing
  • 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_J364003
  • Test Device 5 ：BBa_J364004
  • Test Device 6 ：BBa_J364005

    • >BBa_I20270 Part-only sequence (919 bp)




    Data

    OD600 Reference Point

    Table 1. OD600 Reference Point.

    tabel
    Fluorescein Standard Curve

    Table 2. Data of Fluorescein Standard Curve.

    tabel
    Normalization

    Table 1. OD600 Reference Point.Table 3. Normalization.

    tabel5
    Cell Measurement

    Table 4. Raw data of OD600 measurement.

    tabel