Difference between revisions of "Team:Toronto/InterLab"

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<li>Purpose of this calibration: To transform absorbance data to a OD<sub>600</sub> measurement, calculate a plate-reader specific (Tecan Infinite 200 Pro) conversion factor for OD<sub>600</sub> from Abs<sub>600</sub> calculated for Ludox CL-X on a mass spectrophotometer.</li>
 
<li>Purpose of this calibration: To transform absorbance data to a OD<sub>600</sub> measurement, calculate a plate-reader specific (Tecan Infinite 200 Pro) conversion factor for OD<sub>600</sub> from Abs<sub>600</sub> calculated for Ludox CL-X on a mass spectrophotometer.</li>
 
<li>Beer-Lambert’s law of absorbance dictates that optical path length plays a fundamental role in determining absorbance: </li>
 
<li>Beer-Lambert’s law of absorbance dictates that optical path length plays a fundamental role in determining absorbance: </li>
 +
<br />
 
<img src="https://static.igem.org/mediawiki/2018/b/b5/T--Toronto--7_25_2018_Interlab_lambert-law.gif" alt="lambert-law">
 
<img src="https://static.igem.org/mediawiki/2018/b/b5/T--Toronto--7_25_2018_Interlab_lambert-law.gif" alt="lambert-law">
 +
<br />
 
<li>This is necessary because cuvettes (used in photometer) have a fixed path optical path length when using light scattering to measure absorbance as opposed to the varying path lengths of wells in a 96-well plate which change as the volume of sample added in them changes.</li>
 
<li>This is necessary because cuvettes (used in photometer) have a fixed path optical path length when using light scattering to measure absorbance as opposed to the varying path lengths of wells in a 96-well plate which change as the volume of sample added in them changes.</li>
 
<li>Results: Cell density readings can thus be converted to OD<sub>600</sub> by multiplying correction factor value, <b>4.138</b>.</li>
 
<li>Results: Cell density readings can thus be converted to OD<sub>600</sub> by multiplying correction factor value, <b>4.138</b>.</li>
 
</ul>
 
</ul>
 +
<br />
  
 
<i>Table1.</i>
 
<i>Table1.</i>
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<p><i>Figure 1.</i> Calibration of particle count to absorbance measured by plate-reader (a) A particle standard curve of Abs<sub>600</sub> for known particle count/100ul measurements. (b) Graphical depiction of logarithmic scaling of particle standard curve in Figure 1.(a).</p>
 
<p><i>Figure 1.</i> Calibration of particle count to absorbance measured by plate-reader (a) A particle standard curve of Abs<sub>600</sub> for known particle count/100ul measurements. (b) Graphical depiction of logarithmic scaling of particle standard curve in Figure 1.(a).</p>
 +
<br />
  
 +
<u><b>Calibration 3: Fluorescein Protocol</b></u>
 +
<ul>
 +
<li>Creation of standard fluorescence curve to compare fluorescence output of plate reader instruments between different teams.</li>
 +
<li>Standard curve of fluorescence for fluorescein concentration will allow us to convert cell readings to fluorescence concentration.</li>
 +
</ul>
 +
 +
(a)
 +
<img src="https://static.igem.org/mediawiki/2018/8/80/T--Toronto--7_25_2018_Interlab_graph2.png" alt="graph2">
 +
 +
(b)
 +
<img src="https://static.igem.org/mediawiki/2018/b/bb/T--Toronto--7_25_2018_Interlab_graph3.png" alt="graph3">
 +
<br />
 +
<p>
 +
<i>Figure 2.</i> Calibration of fluorescence measurements to fluorescein concentration  (a) Standard curve of fluorescein fluorescence detected by plate reader
 +
(b) Graphical depiction of logarithmic scaling of <i>Figure 2.(a)</i>.
 +
</p>
 +
<br />
 +
 +
<u><b>Cell Measurement</u></b>
 +
<ul>
 +
<li>transform <i>Escherichia coli</i>DH5α with these following plasmids (all in pSB1C3):
 +
<ul>
 +
  <li>BBa_R0040</li>
 +
  <li>BBa_I20270</li>
 +
  <li>BBa_J364000</li>
 +
  <li>BBa_J364001</li>
 +
  <li>BBa_J364002</li>
 +
  <li>BBa_J364007</li>
 +
  <li>BBa_J364008</li>
 +
  <li>BBa_J364009</li>
 +
</ul></li>
 +
<li>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.</li>
 +
<li>Cell growth, sampling and assay
 +
<ul>
 +
<li>Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of
 +
culture into 4.5mL of LB+Chlor)</li>
 +
<li>Measure Abs<sub>600</sub> of these 1:10 diluted cultures</li>
 +
<li>Dilute the cultures further to a target Abs600 of 0.02 in a final volume of <b>12ml</b> LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light).</li>
 +
<li>Take 500 μL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes,
 +
prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 eppendorf tubes with 500 μL samples per time point, 32 samples total). Place the samples on ice.</li>
 +
<li>Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours.</li>
 +
<li>Take 500 μL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf
 +
tubes. Place samples on ice.</li>
 +
<li>At the end of sampling point,  measure your samples (Abs600 and fluorescence measurement).</li>
 +
</ul>
 +
</li>
 +
</ul>
 +
 +
<img src="https://static.igem.org/mediawiki/2018/7/7c/T--Toronto--7_25_2018_Interlab_picture.png" alt="picture">
 +
<p>“IGEM 2018 InterLab Study Protocol.” <i>IGEM 2018</i>, IGEM Foundation, 2018, 2018.igem.org/wiki/images/0/09/2018_InterLab_Plate_Reader_Protocol.pdf.</p>
 +
<br />
 +
<br />
 +
 +
<u><b>Layout for Fluorescence and Abs<sub>600</sub> measurement</b></u>
 +
<ul>
 +
<li>Two plates as organized below.</li>
 +
<li>One plate for 0 hours, another for 6 hours.</li>
 +
<li>For each plate, measurements of both fluorescence and absorbance will be taken.</li>
 +
</ul>
 +
<br />
 +
<img src="https://static.igem.org/mediawiki/2018/9/9c/T--Toronto--7_25_2018_Interlab_picture1.png" alt="picture1">
 +
<p>“IGEM 2018 InterLab Study Protocol.” <i>IGEM 2018</i>, IGEM Foundation, 2018, 2018.igem.org/wiki/images/0/09/2018_InterLab_Plate_Reader_Protocol.pdf.</p>
  
 
<img src="https://static.igem.org/mediawiki/2018/b/b7/T--Toronto--7_25_2018_Interlab_Capture.png" alt="Capture">
 
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<img src="https://static.igem.org/mediawiki/2018/8/80/T--Toronto--7_25_2018_Interlab_graph2.png" alt="graph2">
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<img src="https://static.igem.org/mediawiki/2018/a/aa/T--Toronto--7_25_2018_Interlab_table3.png" alt="table3">
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<img src="https://static.igem.org/mediawiki/2018/b/bb/T--Toronto--7_25_2018_Interlab_graph3.png" alt="graph3">
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<img src="https://static.igem.org/mediawiki/2018/7/7c/T--Toronto--7_25_2018_Interlab_picture.png" alt="picture">
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<img src="https://static.igem.org/mediawiki/2018/9/9c/T--Toronto--7_25_2018_Interlab_picture1.png" alt="picture1">
 
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<img src="https://static.igem.org/mediawiki/2018/a/a8/T--Toronto--7_25_2018_Interlab_picture2.png" alt="picture2">
 
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</body>
 
</body>
  

Revision as of 01:54, 26 July 2018

INTERLAB

Calibration 1: OD600 Reference point
  • Purpose of this calibration: To transform absorbance data to a OD600 measurement, calculate a plate-reader specific (Tecan Infinite 200 Pro) conversion factor for OD600 from Abs600 calculated for Ludox CL-X on a mass spectrophotometer.
  • Beer-Lambert’s law of absorbance dictates that optical path length plays a fundamental role in determining absorbance:

    • lambert-law
  • This is necessary because cuvettes (used in photometer) have a fixed path optical path length when using light scattering to measure absorbance as opposed to the varying path lengths of wells in a 96-well plate which change as the volume of sample added in them changes.
  • Results: Cell density readings can thus be converted to OD600 by multiplying correction factor value, 4.138.

Table1.

Table shows absorbance measurements (at 600 nm) for LUDOX CL-X and dd-H2O using a plate reader. The corrected Abs600 is the difference between the LUDOX CL-X reading and dd H2O reading. Reference OD600 is a measurement by a spectrophotometer (provided on iGEM excel sheet). OD600/Abs600 is the correction factor to convert Abs600 to OD600, calculated by dividing Reference OD600 by Abs600.

LUDOX_CL_X

Calibration 2: Particle Standard Curve

Construct a standard curve of Abs600 for microsphere particle concentration Purpose of calibration: iGEM distributed microspheres that mimic the size, shape and volume of cells which have a known amount of microspheres per volume. This calibration was required to generate a Particle Standard Curve which helped us determine the number of cells (as modelled by microspheres) in a sample.

  • Use standard curve to convert Abs600 measurements to an estimate of number of cells.
  • Monodisperse silica microspheres are used in the calibration because they have similar size and optical characteristics as cells.
graph graph1

Figure 1. Calibration of particle count to absorbance measured by plate-reader (a) A particle standard curve of Abs600 for known particle count/100ul measurements. (b) Graphical depiction of logarithmic scaling of particle standard curve in Figure 1.(a).


Calibration 3: Fluorescein Protocol
  • Creation of standard fluorescence curve to compare fluorescence output of plate reader instruments between different teams.
  • Standard curve of fluorescence for fluorescein concentration will allow us to convert cell readings to fluorescence concentration.
(a) graph2 (b) graph3

Figure 2. Calibration of fluorescence measurements to fluorescein concentration (a) Standard curve of fluorescein fluorescence detected by plate reader (b) Graphical depiction of logarithmic scaling of Figure 2.(a).


Cell Measurement
  • transform Escherichia coliDH5α with these following plasmids (all in pSB1C3):
    • BBa_R0040
    • BBa_I20270
    • BBa_J364000
    • BBa_J364001
    • BBa_J364002
    • BBa_J364007
    • BBa_J364008
    • BBa_J364009
  • 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.
  • Cell growth, sampling and assay
    • Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)
    • Measure Abs600 of these 1:10 diluted cultures
    • Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12ml LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light).
    • Take 500 μL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 eppendorf tubes with 500 μL samples per time point, 32 samples total). Place the samples on ice.
    • Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours.
    • Take 500 μL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes. Place samples on ice.
    • At the end of sampling point, measure your samples (Abs600 and fluorescence measurement).
picture

“IGEM 2018 InterLab Study Protocol.” IGEM 2018, IGEM Foundation, 2018, 2018.igem.org/wiki/images/0/09/2018_InterLab_Plate_Reader_Protocol.pdf.



Layout for Fluorescence and Abs600 measurement
  • Two plates as organized below.
  • One plate for 0 hours, another for 6 hours.
  • For each plate, measurements of both fluorescence and absorbance will be taken.

picture1

“IGEM 2018 InterLab Study Protocol.” IGEM 2018, IGEM Foundation, 2018, 2018.igem.org/wiki/images/0/09/2018_InterLab_Plate_Reader_Protocol.pdf.

Capture Capture1 Capture2 Capture3 Capture4 table2 table3 picture1 picture2