Difference between revisions of "Team:DLUT China B/InterLab"

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    <title>InterLab</title>
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        <div id="background" class="interLab"></div>
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        <div class="moduleTitle">InterLab</div>
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    </div>
  
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        <div id="contents"></div>
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        <div id="article">
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            <div class="mainText">
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                <p>The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability
 +
                    in synthetic biology measurements, so that eventually, measurements that are taken in different labs
 +
                    will be no more variable than measurements taken within the same lab. </p>
 +
                <p>This year, we use two orthogonal approaches to compute the cell count in our samples.</p>
 +
                <ol>
 +
                    <li>Converting between absorbance of cells to absorbance of a known concentration of beads.</li>
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                    <li>Counting colony-forming units (CFUs) from the sample.</li>
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                </ol>
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            </div>
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            <div>
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                <h2 class="title">Plate reader and CFU</h2>
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                <div class="mainText">
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                    <h3 class="title">Calibration protocol</h3>
 +
                    <p>Before we started the plate reader measurement, we obtained the OD600 reference point, the
 +
                        fluorescein fluorescence curve and the particle standard curve in the microplate reader to
 +
                        standardize the absorbance reading, cell-based fluorescence reading and the number of
 +
                        cells.</p>
 +
                    <p>Our model was a SpectraMax M2e Plate Reader. For the plate reader our excitation and emission
 +
                        settings were 485 nm and 525 nm respectively (Same setting was used for all experiments
 +
                        below).</p>
 +
                    <h3 class="title">Cell measurement protocol</h3>
 +
                    <p>Transformation</p>
 +
                    <p>We transformed the plasmids (listed below) resuspended from the Distribution Kit into E. coli
 +
                        DH5-alpha cells. Colonies were given 16 hours to grow.</p>
 +
                    <div class="img"><img src="https://static.igem.org/mediawiki/2018/8/81/T--DLUT_China_B--InterLab1.png"></div>
 +
                    <p>2 colonies of each device were inoculated over night into 5 ml Luria-Bertani media with 25 μg/mL
 +
                        Chloramphenicol in a 37°C, 220 rpm shaking incubator. Cell cultures were diluted to a target
 +
                        OD600 of 0.02 into same LB medium in 50 mL falcon tube covered with foil before use Diluted
 +
                        cultures were further grown at 37°C and 220 rpm. At 0, and 6 hours of incubation, 500 μL aliquot
 +
                        was taken from each two colonies of the 8 devices and were placed immediately on ice to prevent
 +
                        further growth. At the end of sampling point, 4 replicates 100 μl of each sample was pipetted
 +
                        into a 96-well microplate with the arrangement. Data was imported into the Excel Sheet for
 +
                        submission.</p>
 +
                    <h3 class="title">Protocol: Colony Forming Units per 0.1OD<sub>600</sub> E.coli cultures</h3>
 +
                    <p>This protocol can be calibrate OD<sub>600</sub> to colony forming unit(CFU) counts, which are
 +
                        directly relatable to the cell concentration of the culture. At this protocol, we first Dilute
 +
                        our overnight culture to OD<sub>600</sub> =0.1in 1ml of LB+Cam media. We further diluted the
 +
                        culture according to the experimental protocol than cultivate these dilutions for 18-20hours.
 +
                        Counting colony-forming units (CFUs) from the sample.</p>
 +
                    <h3 class="title">Results and Discussion</h3>
 +
                    <p>Below is the Fluorescein Standard Curve and the particle standard curve we obtained.</p>
 +
                    <div class="img"><img src="https://static.igem.org/mediawiki/2018/7/77/T--DLUT_China_B--InterLab2.png"></div>
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                    <div class="img"><img src="https://static.igem.org/mediawiki/2018/d/d5/T--DLUT_China_B--InterLab3.png"></div>
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                    <div class="img"><img src="https://static.igem.org/mediawiki/2018/b/b5/T--DLUT_China_B--InterLab4.png"></div>
 +
                    <p>In the Fluorescein Standard Curve, one of the four sets of data we measured had a problem with
 +
                        dilution.</p>
 +
                    <p>Below is Cell measurement protocol.</p>
 +
                    <table class="img">
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                        <tr>
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                            <td><img src="https://static.igem.org/mediawiki/2018/0/02/T--DLUT_China_B--InterLab5.png"></td>
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                            <td><img src="https://static.igem.org/mediawiki/2018/a/a7/T--DLUT_China_B--InterLab6.png"></td>
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                        </tr>
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                    </table>
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                </div>
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            </div>
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            <div>
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                <h2 class="title">Flow Cytome Protocol</h2>
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                <div class="mainText">
 +
                    <p>In addition, we also done the Flow Cytome Protocol. We used a flow cytometer and SpheroTech
 +
                        calibration beads collected and submitted flow cytometry data. But our flow cytometer cannot be
 +
                        directly measured using 96 well plate, so we only measured A1-A10, E1-E9 for each time. As for
 +
                        well A9 and E9, we used the same blank media - 200 μL of LB + Cam for each time. Below is some
 +
                        results.</p>
 +
                    <table class="img">
 +
                        <tr>
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                            <td><img src="https://static.igem.org/mediawiki/2018/e/ee/T--DLUT_China_B--InterLab7.png"></td>
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                            <td><img src="https://static.igem.org/mediawiki/2018/8/88/T--DLUT_China_B--InterLab8.png"></td>
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                        </tr>
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                        <tr>
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                            <td><img src="https://static.igem.org/mediawiki/2018/f/f7/T--DLUT_China_B--InterLab9.png"></td>
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                            <td><img src="https://static.igem.org/mediawiki/2018/5/54/T--DLUT_China_B--InterLab10.png"></td>
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                        </tr>
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                        <tr>
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                            <td><img src="https://static.igem.org/mediawiki/2018/5/50/T--DLUT_China_B--InterLab11.png"></td>
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                            <td><img src="https://static.igem.org/mediawiki/2018/1/10/T--DLUT_China_B--InterLab12.png"></td>
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                        </tr>
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                    </table>
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                    <p>SpheroTech calibration beads</p>
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Latest revision as of 08:40, 17 October 2018

InterLab

InterLab

The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab. 

This year, we use two orthogonal approaches to compute the cell count in our samples.

  1. Converting between absorbance of cells to absorbance of a known concentration of beads.
  2. Counting colony-forming units (CFUs) from the sample.

Plate reader and CFU

Calibration protocol

Before we started the plate reader measurement, we obtained the OD600 reference point, the fluorescein fluorescence curve and the particle standard curve in the microplate reader to standardize the absorbance reading, cell-based fluorescence reading and the number of cells.

Our model was a SpectraMax M2e Plate Reader. For the plate reader our excitation and emission settings were 485 nm and 525 nm respectively (Same setting was used for all experiments below).

Cell measurement protocol

Transformation

We transformed the plasmids (listed below) resuspended from the Distribution Kit into E. coli DH5-alpha cells. Colonies were given 16 hours to grow.

2 colonies of each device were inoculated over night into 5 ml Luria-Bertani media with 25 μg/mL Chloramphenicol in a 37°C, 220 rpm shaking incubator. Cell cultures were diluted to a target OD600 of 0.02 into same LB medium in 50 mL falcon tube covered with foil before use Diluted cultures were further grown at 37°C and 220 rpm. At 0, and 6 hours of incubation, 500 μL aliquot was taken from each two colonies of the 8 devices and were placed immediately on ice to prevent further growth. At the end of sampling point, 4 replicates 100 μl of each sample was pipetted into a 96-well microplate with the arrangement. Data was imported into the Excel Sheet for submission.

Protocol: Colony Forming Units per 0.1OD600 E.coli cultures

This protocol can be calibrate OD600 to colony forming unit(CFU) counts, which are directly relatable to the cell concentration of the culture. At this protocol, we first Dilute our overnight culture to OD600 =0.1in 1ml of LB+Cam media. We further diluted the culture according to the experimental protocol than cultivate these dilutions for 18-20hours. Counting colony-forming units (CFUs) from the sample.

Results and Discussion

Below is the Fluorescein Standard Curve and the particle standard curve we obtained.

In the Fluorescein Standard Curve, one of the four sets of data we measured had a problem with dilution.

Below is Cell measurement protocol.

Flow Cytome Protocol

In addition, we also done the Flow Cytome Protocol. We used a flow cytometer and SpheroTech calibration beads collected and submitted flow cytometry data. But our flow cytometer cannot be directly measured using 96 well plate, so we only measured A1-A10, E1-E9 for each time. As for well A9 and E9, we used the same blank media - 200 μL of LB + Cam for each time. Below is some results.

SpheroTech calibration beads