Difference between revisions of "Team:BIT/InterLab"

 
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            <h1>Interlab</h1>
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            <p>The goal of the iGEM InterLab Study is to improve the measurement tools available to both the iGEM community and the synthetic biology community as. In the previous studies, we showed that by measuring GFP expression in absolute fluorescence units calibrated against a known concentration of fluorescent molecule, we can greatly reduce the variability in measurements between labs. while measuring a population of cells ,the number of cells in the sample can lead to variability in the results.
 +
            </p>
 +
            <p><b>So,the aim of the fifth InterLab study is trying to reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD.
 +
            </b></p>
 +
            <p>
 +
                We performed three calibration experiments and cell measurements, including flow cytometry.<br>
 +
                <b>Calibration 1: OD600 Reference point - LUDOX Protocol</b><br>
 +
                Using LUDOX CL-X and H2O as point reference to obtain a conversion factor to transform the data later.The average of OD600 is 0.053;the correction factor (OD600/Abs600) is 3.761.
 +
            </p>
 +
            <img src="https://static.igem.org/mediawiki/2018/0/0b/T--BIT--iGEM_BIT2018_INTERLAB_WORK_1.png">
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            <span>Table1,OD600 Reference point</span>
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            <p>
 +
                <b>Calibration 2:Particle Standard Curve - Microsphere Protocol</b><br>
 +
                Preparing a dilution series of monodisperse silica microspheres and measure the Abs 600 to abtain the particle standard curve.
 +
            </p>
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            <div class="half">
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                <div class="half1">
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                    <img src="https://static.igem.org/mediawiki/2018/3/37/T--BIT--iGEM_BIT2018_INTERLAB_WORK_2.png">
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                    <span>Figure1,Particle Standard Curve</span>
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                </div>
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                <div class="half2">
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                    <img src="https://static.igem.org/mediawiki/2018/3/3e/T--BIT--iGEM_BIT2018_INTERLAB_WORK_3.png">
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                    <span>Figure2,Particle Standard Curve(log scale)</span>
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                </div>
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            </div>
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            <p>
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                <b>Calibration 3: Fluorescence standard curve - Fluorescein Protocol</b><br>
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                Dilution serious of fluorescein were prepared in four replicates and measure the fluorescence in a 96 well plate,so the standard curve of fluorescence for fluorescein concentration is generated.
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            </p>
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            <div class="half">
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                <div class="half1">
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                    <img src="https://static.igem.org/mediawiki/2018/0/0e/T--BIT--iGEM_BIT2018_INTERLAB_WORK_4.png">
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                    <span>Figure3,Fluorescein Standard Curve</span>
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                </div>
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                <div class="half2">
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                    <img src="https://static.igem.org/mediawiki/2018/5/58/T--BIT--iGEM_BIT2018_INTRELAB_WORK_5.png">
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                    <span>Figure4,Fluorescein Standard Curve(log scale)</span>
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                </div>
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            </div>
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            <h2>Cell measurement</h2>
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            <p>
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                1.Using E. coli K-12 DH5-alpha to do the Cell measurement, detecting the Fluorescence and Abs600.
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            </p>
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            <img src="https://static.igem.org/mediawiki/2018/5/58/T--BIT--iGEM_BIT2018_INTERLAB_WORK_11.png">
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            <span>Figure5,The workflow of cell measurement</span>
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            <h3>Here is our experimental data:</h3>
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            <img src="https://static.igem.org/mediawiki/2018/d/df/T--BIT--iGEM_BIT2018_INTERLAB_4.1.1.jpg">
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            <h4>Table2,The Fluorescence Raw Reading of 0 Hour</h4>
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            <img src="https://static.igem.org/mediawiki/2018/c/c0/T--BIT--iGEM_BIT2018_INTERLAB_WORK_7.png">
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            <h4>Table3,The Fluorescence Raw Reading of 6 Hour</h4>
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            <img src="https://static.igem.org/mediawiki/2018/3/3a/T--BIT--iGEM_BIT2018_INTERLAB_WORK_8.png">
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            <h4>Table4,The Abs Raw Reading of 0 Hour</h4>
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            <img src="https://static.igem.org/mediawiki/2018/0/04/T--BIT--iGEM_BIT2018_INTERLAB_WORK_9.png">
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            <h4>Table5,The Abs Raw Reading of 6 Hour</h4>
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            <p>2.The measurement of Colony Forming Units per 0.1 OD600 E. coli cultures.</p>
 +
            <img src="https://static.igem.org/mediawiki/2018/3/30/T--BIT--iGEM_BIT2018_INTERLAB_WORK_12.png">
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            <span>Figure6,The workflow of  Dilution Series</span>
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            <p>The following is our experimental data. Since we forgot to dilute the solution ten times at the end, only two diluents with concentration gradient are effective.
 +
            </p>
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            <img src="https://static.igem.org/mediawiki/2018/7/7b/T--BIT--iGEM_BIT2018_INTRELAB_WORK_10.png">
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            <h4>Table6,The number of colonies</h4>
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            <h3>Here are pictures of our E.coli culture:</h3>
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            <div id="six-pic">
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                <div class="three-pic">
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                    <img src="https://static.igem.org/mediawiki/2018/d/d8/T--BIT--InterLab_Figure-CFU1.jpeg">
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                    <img src="https://static.igem.org/mediawiki/2018/1/1c/T--BIT--InterLab_Figure_CFU2.jpeg">
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                    <img src="https://static.igem.org/mediawiki/2018/8/80/T--BIT--InterLab_Figure_CFU3.jpeg">
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                </div>
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                <div class="three-pic">
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                    <img src="https://static.igem.org/mediawiki/2018/5/55/T--BIT--InterLab_Figure-CFU4.jpeg">
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                    <img src="https://static.igem.org/mediawiki/2018/0/07/T--BIT--InterLab_Figure-CFU5.jpeg">
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                    <img src="https://static.igem.org/mediawiki/2018/8/88/T--BIT--InterLab_Figure-CFU6.jpeg">
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                </div>
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            </div>
 +
            <h2>Flow Cytometry</h2>
 +
            <p>Here are some of our results:</p>
 +
            <div class="half">
 +
                <div class="half1">
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                    <img src="https://static.igem.org/mediawiki/2018/b/b6/T--BIT--iGEM_BIT2018_LNTERLAB_WORK_13.png">
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                    <span>Figure7,The negative control</span>
 +
                </div>
 +
                <div class="half2">
 +
                    <img src="https://static.igem.org/mediawiki/2018/e/ef/T--BIT--iGEM_BIT2018_INTERLAB_WORK_14.png">
 +
                    <span>Figure8,The positive control</span>
 +
                </div>
 +
            </div>
 +
            <p>The closer to the left, the less fluorescence  The closer to the right, the more fluorescence</p>
 +
            <div class="half">
 +
                <div class="half1">
 +
                    <img src="https://static.igem.org/mediawiki/2018/1/1f/T--BIT--iGEM_BIT2018_INTERLAB_WORK_18.png">
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                    <span>Figure9,Resule of BBa_j364001</span>
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                </div>
 +
                <div class="half2">
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                    <img src="https://static.igem.org/mediawiki/2018/8/8b/T--BIT--iGEM_BIT2018_INTERLAB_WORK_19.png">
 +
                    <span> Figure10,Result of BBa_j364002</span>
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                </div>
 +
            </div>
 +
            <p>For more results, click here</p>
 +
            <p>The following is a diagram of the parallel experimental results of the SpheroTech Rainbow calibration beads, and eight peaks can be clearly seen.
 +
            </p>
 +
            <img src="https://static.igem.org/mediawiki/2018/f/f3/T--BIT--iGEM_BIT2018_LNTERLAB_WORK_15.png">
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        </div>
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    </div>
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<h1>OD<sub>600</sub> Reference point</h1>
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<b>Materia</b>
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<br>
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1ml LUDOX CL-X (provided in kit)<br>
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ddH​ 2​ 0 (provided by team)<br>
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96 well plate, black with clear flat bottom preferred (provided by team)<br>
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<br><br>
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<b>Method</b>
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<br>
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Add 100 μl LUDOX into wells A1, B1, C1, D1<br>
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Add 100 μl of dd H​ 2​ O into wells A2, B2, C2, D2<br>
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Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for
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cell measurements<br>
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Record the data in the table below or in your notebook<br>
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Import data into Excel sheet provided (​OD<sub>600</sub> reference point tab​​)<br>
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<br>
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<b>Results</b>
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<br>
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<br><br>
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<h1>  Particle Standard Curve </h1>
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<p>
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<b>Materia</b>
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<br>
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300 μL Silica beads - Microsphere suspension (provided in kit, 4.7 x 10^8 microspheres)<br>
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ddH20 (provided by team) 96 well plate, black with clear flat bottom preferred (provided by team)
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<br><br>
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<b>Method</b>
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<br>
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<b>Prepare:</b><br>
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Stock Solution<br>
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Obtain the tube labeled “Silica Beads” from the InterLab test kit and vortex<br>
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    Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube<br>
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vigorously for 30 seconds.<br>
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Add 904 μL of ddH2O to the microspheres<br>
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Vortex well. <br>
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<b>serial dilution:</b><br>
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Add 100 μl of ddH20 into wells A2, B2, C2, D2....A12, B12, C12, D12<br>
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Vortex the tube containing the stock solution of microspheres vigorously for 10 seconds<br>
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Immediately add 200 μlof microspheres stock solution into A1<br>
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Transfer 100 μl of microsphere stock solution from A1 into A2.<br>
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Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…<br>
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Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...<br>
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Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...<br>
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Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...<br>
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Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...<br>
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Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...<br>
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Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...<br>
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Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...<br>
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Mix A10 by pipetting up and down 3x and transfer 100 μl into A11...<br>
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Mix A11 by pipetting up and down 3x and transfer 100 μl into ​liquid waste<br>
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<br>
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<b>Results</b>
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<br>
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<br><br>
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<h1>The standard curve of Fluorescein's fluorescence</h1>
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<p>
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<b>Materia</b>
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<br>
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Fluorescein (provided in kit)<br>
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10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team)<br>
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96 well plate, black with clear flat bottom (provided by team)<br>
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<br>
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<b>Method</b>
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<br>
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<b>Prepare the fluorescein stock solution:</b><br>
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Spin down fluorescein kit tube to make sure pellet is at the bottom of tube.<br>
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Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1
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mL of 1xPBS<br>
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Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein<br>
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Solution with concentration 10 μM: 100 μL of 10x fluorescein stock into 900 μL 1x
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PBS<br>
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<b>serial dilutions:</b><br>
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Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12<br>
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Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1<br>
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Transfer 100 μl of fluorescein stock solution from A1 into A2.<br>
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Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…<br>
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Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...<br>
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Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...<br>
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Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...<br>
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Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...<br>
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Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...<br>
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Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...<br>
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Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...<br>
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Mix A10 by pipetting up and down 3x and transfer 100 μl into A11...<br>
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Mix A11 by pipetting up and down 3x and transfer 100 μl into ​liquid waste<br>
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Repeat dilution series for rows B, C, D<br>
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<br>
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<b>Results</b>
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<br>
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<br><br>
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</p>
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<h1>Cell measure</h1>
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<p>
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<b>Materia</b><br>
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Competent cells (​Escherichia coli strain DH5α)<br>
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LB (Luria Bertani) media<br>
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Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)<br>
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50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)<br>
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Incubator at 37°C<br>
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1.5 ml eppendorf tubes for sample storage<br>
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Ice bucket with ice<br>
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Micropipettes and tips<br>
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96 well plate, black with clear flat bottom preferred (provided by team)<br>
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<br>
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<b>Method</b>
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<br>
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Day 1​​: transform ​Escherichia coli DH5α with these following plasmids (all in pSB1C3) <br>
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  Day 2​​: Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.<br>
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Day 3 and after​​: Cell growth, sampling, and assay<br>
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1、Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of
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culture into 4.5mL of LB+Chlor)<br>
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2、Measure Abs​ 600​ of these 1:10 diluted cultures<br>
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3、Record the data in the notebook<br>
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4、 Dilute the cultures further to a target Abs​ 600 of 0.02 in a final volume of ​12 ml LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block
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light).<br>
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5、Take 500 µL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes,
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prior to incubation.Place the samples on ice.<br>
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6、Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours.<br>
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7、 Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf
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tubes. Place samples on ice.<br>
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8、At the end of sampling point you need to measure your samples (Abs​ 600 and
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fluorescence measurement), see the below for details.<br><br>
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<b>Results</b>
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<br>
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<img src="https://static.igem.org/mediawiki/2018/d/d8/T--BIT--InterLab_Figure-CFU1.jpeg" width="233" height="166"  class="img-fluid tm-img"><br>
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<img src="https://static.igem.org/mediawiki/2018/1/1c/T--BIT--InterLab_Figure_CFU2.jpeg" width="233" height="166"  class="img-fluid tm-img"><br>
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<img src="https://static.igem.org/mediawiki/2018/8/80/T--BIT--InterLab_Figure_CFU3.jpeg" width="233" height="166"  class="img-fluid tm-img"><br>
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<img src="https://static.igem.org/mediawiki/2018/5/55/T--BIT--InterLab_Figure-CFU4.jpeg" width="233" height="166"  class="img-fluid tm-img"><br>
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<img src="https://static.igem.org/mediawiki/2018/0/07/T--BIT--InterLab_Figure-CFU5.jpeg" width="233" height="166"  class="img-fluid tm-img"><br>
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<img src="https://static.igem.org/mediawiki/2018/8/88/T--BIT--InterLab_Figure-CFU6.jpeg" width="233" height="166"  class="img-fluid tm-img"><br>
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Latest revision as of 00:56, 18 October 2018

<!DOCTYPE html> Interlab

Interlab

The goal of the iGEM InterLab Study is to improve the measurement tools available to both the iGEM community and the synthetic biology community as. In the previous studies, we showed that by measuring GFP expression in absolute fluorescence units calibrated against a known concentration of fluorescent molecule, we can greatly reduce the variability in measurements between labs. while measuring a population of cells ,the number of cells in the sample can lead to variability in the results.

So,the aim of the fifth InterLab study is trying to reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD.

We performed three calibration experiments and cell measurements, including flow cytometry.
Calibration 1: OD600 Reference point - LUDOX Protocol
Using LUDOX CL-X and H2O as point reference to obtain a conversion factor to transform the data later.The average of OD600 is 0.053;the correction factor (OD600/Abs600) is 3.761.

Table1,OD600 Reference point

Calibration 2:Particle Standard Curve - Microsphere Protocol
Preparing a dilution series of monodisperse silica microspheres and measure the Abs 600 to abtain the particle standard curve.

Figure1,Particle Standard Curve
Figure2,Particle Standard Curve(log scale)

Calibration 3: Fluorescence standard curve - Fluorescein Protocol
Dilution serious of fluorescein were prepared in four replicates and measure the fluorescence in a 96 well plate,so the standard curve of fluorescence for fluorescein concentration is generated.

Figure3,Fluorescein Standard Curve
Figure4,Fluorescein Standard Curve(log scale)

Cell measurement

1.Using E. coli K-12 DH5-alpha to do the Cell measurement, detecting the Fluorescence and Abs600.

Figure5,The workflow of cell measurement

Here is our experimental data:

Table2,The Fluorescence Raw Reading of 0 Hour

Table3,The Fluorescence Raw Reading of 6 Hour

Table4,The Abs Raw Reading of 0 Hour

Table5,The Abs Raw Reading of 6 Hour

2.The measurement of Colony Forming Units per 0.1 OD600 E. coli cultures.

Figure6,The workflow of Dilution Series

The following is our experimental data. Since we forgot to dilute the solution ten times at the end, only two diluents with concentration gradient are effective.

Table6,The number of colonies

Here are pictures of our E.coli culture:

Flow Cytometry

Here are some of our results:

Figure7,The negative control
Figure8,The positive control

The closer to the left, the less fluorescence The closer to the right, the more fluorescence

Figure9,Resule of BBa_j364001
Figure10,Result of BBa_j364002

For more results, click here

The following is a diagram of the parallel experimental results of the SpheroTech Rainbow calibration beads, and eight peaks can be clearly seen.