Difference between revisions of "Team:McMasterU/InterLab"

 
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<h1>Overview</h1>
 
<h1>Overview</h1>
 
<p>The Interlab Study, developed by the Measurement Committee of iGEM as a way to standardize measurements for green fluorescent protein (GFP), tackles the foundational synthetic biology problem of a lack of reliable measurements for experiments. With an international standard set down, scientists can go beyond utilizing relativity comparisons for results, to conduct quantitative reproducibility studies across labs. This Interlab experiment, carried out by McMaster iGEM with plate readers, aims to produce comparable GFP measurements as a contribution for the international standardization of GFP values. The parts we used were BBA_J364000 (1), BBa_J364001 (2), BBa_J364002 (3), BBa_J36003 (4), BBa_J364004 (5), BBa_J364005 (6), and positive control BBa_120270 with negative control BBa_R0040. All parts were located in pSB1C3 plasmid and were modified for chloramphenicol resistance. <br /><br /></p>
 
<p>The Interlab Study, developed by the Measurement Committee of iGEM as a way to standardize measurements for green fluorescent protein (GFP), tackles the foundational synthetic biology problem of a lack of reliable measurements for experiments. With an international standard set down, scientists can go beyond utilizing relativity comparisons for results, to conduct quantitative reproducibility studies across labs. This Interlab experiment, carried out by McMaster iGEM with plate readers, aims to produce comparable GFP measurements as a contribution for the international standardization of GFP values. The parts we used were BBA_J364000 (1), BBa_J364001 (2), BBa_J364002 (3), BBa_J36003 (4), BBa_J364004 (5), BBa_J364005 (6), and positive control BBa_120270 with negative control BBa_R0040. All parts were located in pSB1C3 plasmid and were modified for chloramphenicol resistance. <br /><br /></p>
 +
 
<h1>Methods and Materials</h1>
 
<h1>Methods and Materials</h1>
 
<h2>Plate Reader</h2>
 
<h2>Plate Reader</h2>
 
<p>Plate Reader: TECAN Infinite M1000 Plate: Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom) <br /> <strong>Abs OD600</strong></p>
 
<p>Plate Reader: TECAN Infinite M1000 Plate: Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom) <br /> <strong>Abs OD600</strong></p>
−
<ul>Wavelengths: 600 nm</ul>
+
<ul>
−
<ul>Number of Flashes: 20</ul>
+
  <li> Wavelengths: 600 nm</li>
−
<ul>Settle Time: 0 ms</ul>
+
  <li>Number of Flashes: 20</li>
−
<ul>Temperature: 26.1 degrees Celsius</ul>
+
  <li>Settle Time: 0 ms</li>
−
<p><br /> <strong>Fluorescence</strong></p>
+
  <li>Temperature: 26.1 degrees Celsius</li>
−
<ul>Excitation:485 nm</ul>
+
</ul>
−
<ul>Emission: 535 nm</ul>
+
  <p><strong>Fluorescence</strong></p>
−
<ul>Excitation Bandwidth: 5 nm</ul>
+
<ul>
−
<ul>Emission Bandwidth: 5 nm</ul>
+
  <li>Excitation:485 nm</li>
−
<ul>Optics: Bottom Reading</ul>
+
  <li>Emission: 535 nm</li>
−
<ul>Gain: Optimal (automatic)</ul>
+
  <li>Excitation Bandwidth: 5 nm</li>
−
<ul>Number of Flashes: 50</ul>
+
  <li>Emission Bandwidth: 5 nm</li>
−
<ul>Flash Frequency: 400 Hz</ul>
+
  <li>Optics: Bottom Reading</li>
−
<ul>Integration Time: 20 us</ul>
+
  <li>Gain: Optimal (automatic)</li>
−
<ul>Lag Time: 0 us</ul>
+
  <li>Number of Flashes: 50</li>
−
<ul>Settle Time: 0 ms</ul>
+
  <li>Flash Frequency: 400 Hz</li>
−
<ul>Part of Plate: A1-H10</ul>
+
  <li>Integration Time: 20 us</li>
−
<ul>Temperature: 26.8 degrees Celsius</ul>
+
  <li>Lag Time: 0 us</li>
 +
  <li>Settle Time: 0 ms</li>
 +
  <li>Part of Plate: A1-H10</li>
 +
  <li>Temperature: 26.8 degrees Celsius</li>
 +
</ul>
 
<p><br /><br /></p>
 
<p><br /><br /></p>
 +
 
<h2>OD600 Reference Point</h2>
 
<h2>OD600 Reference Point</h2>
 
<p><strong>Materials</strong></p>
 
<p><strong>Materials</strong></p>
−
<ul>1 mL LUDOX (provided for by iGEM Headquarters)</ul>
+
<ul>
−
<ul>Distilled H2O</ul>
+
  <li>1 mL LUDOX (provided for by iGEM Headquarters)</li>
−
<ul>96 well plate, Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)</ul>
+
  <li>Distilled H2O</li>
 +
  <li>96 well plate, Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)</li>
 +
</ul>
 +
 
 
<p><br /> <strong>Methods</strong></p>
 
<p><br /> <strong>Methods</strong></p>
 
<ol>
 
<ol>
−
<li>Add 100 uL of LUDOX into A1, B1, C1, and D1 wells</li>
+
  <li>Add 100 uL of LUDOX into A1, B1, C1, and D1 wells</li>
−
<li>Add 1 mL LUDOX into a separate cuvette</li>
+
  <li>Add 1 mL LUDOX into a separate cuvette</li>
−
<li>Add 100 uL of dH2O into wells A2, B2, C2, D2 wells</li>
+
  <li>Add 100 uL of dH2O into wells A2, B2, C2, D2 wells</li>
−
<li>Add 1 mL dH2O into a separate cuvette</li>
+
  <li>Add 1 mL dH2O into a separate cuvette</li>
−
<li>Image using plate reader (TECAN Infinite M1000)</li>
+
  <li>Image using plate reader (TECAN Infinite M1000)</li>
 
</ol>
 
</ol>
 +
 
<p><br /><br /></p>
 
<p><br /><br /></p>
 +
 
<h2>Protocol of Fluorescein Fluorescence Standard Curve</h2>
 
<h2>Protocol of Fluorescein Fluorescence Standard Curve</h2>
 
<p><strong>Materials:</strong></p>
 
<p><strong>Materials:</strong></p>
 
<ul>
 
<ul>
−
<li>Fluorescein</li>
+
  <li>Fluorescein</li>
−
<li>10 mL 1x PBS (phosphate buffered saline)</li>
+
  <li>10 mL 1x PBS (phosphate buffered saline)</li>
−
<li>96 well plate, Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)</li>
+
  <li>96 well plate, Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)</li>
 
</ul>
 
</ul>
 +
 
<p><br /> <strong>Methods:</strong></p>
 
<p><br /> <strong>Methods:</strong></p>
 
<ol>
 
<ol>
−
<li>Spin down fluorescence stock tube until pellet is at bottom of tube</li>
+
  <li>Spin down fluorescence stock tube until pellet is at bottom of tube</li>
−
<li>Resuspend 100 uM stock Fluorescein in 1 mL of 1x PBS to obtain 2x fluorescein stock solution.</li>
+
  <li>Resuspend 100 uM stock Fluorescein in 1 mL of 1x PBS to obtain 2x fluorescein stock solution.</li>
−
<li>Ensure that Fluorescein is properly dissolved.</li>
+
  <li>Ensure that Fluorescein is properly dissolved.</li>
−
<li>Dilute 2x fluorescin stock solution with 1x PBS to make a 1x fluorescein solution. Resulting concentration of fluorescein stock solution should be 50 uM (otherwise known as 500 uL of 2x fluoresceine in 500 uL of 1x PBS that makes 1 mL of 50 uM of 1x fluorescein solution).</li>
+
  <li>Dilute 2x fluorescin stock solution with 1x PBS to make a 1x fluorescein solution. Resulting concentration of fluorescein stock solution should be 50 uM (otherwise known as 500 uL of 2x fluoresceine in 500 uL of 1x PBS that makes 1 mL of 50 uM of 1x fluorescein solution).</li>
−
<li>Prepare for serial dilutions across columns 1 to 11. Ensure column 12 has PBS buffer only.
+
  <li>Prepare for serial dilutions across columns 1 to 11. Ensure column 12 has PBS buffer only.
−
<ul>
+
  <ul>
−
<li>Pipette 200 uL of 1x fluorescein stock into wells A1, B1, C1, D1.</li>
+
      <li>Pipette 200 uL of 1x fluorescein stock into wells A1, B1, C1, D1.</li>
−
<li>Pipette 100 uL of PBS in wells A2, B2, C2, D2 ... A12, B12.</li>
+
      <li>Pipette 100 uL of PBS in wells A2, B2, C2, D2 ... A12, B12.</li>
−
<li>Transfer 100 uL of fluorescein stock solution from A1 to A2.</li>
+
      <li>Transfer 100 uL of fluorescein stock solution from A1 to A2.</li>
−
<li>Mix A2 by pipetting up and down 3x and transfer 100 uL into A3.</li>
+
      <li>Mix A2 by pipetting up and down 3x and transfer 100 uL into A3.</li>
−
<li>Mix A3 by pipetting up and down 3x and transfer 100 uL into A4.</li>
+
      <li>Mix A3 by pipetting up and down 3x and transfer 100 uL into A4.</li>
−
<li>Repeat 5c to 5e for A4, A5, A6.... to A11.</li>
+
      <li>Repeat 5c to 5e for A4, A5, A6.... to A11.</li>
−
<li>Mix A11 by pipetting up and down 3x and then transfer 100 uL into liquid waste. Ensure that A12 is PBS only</li>
+
      <li>Mix A11 by pipetting up and down 3x and then transfer 100 uL into liquid waste. Ensure that A12 is PBS only</li>
−
</ul>
+
  </ul>
−
</li>
+
  </li>
−
<li>Repeat step 5 for columns B, C, and D.</li>
+
  <li>Repeat step 5 for columns B, C, and D.</li>
−
<li>Measure fluorescence of all samples in the plate reader (settings listed above in Plate Reader Setup menu).</li>
+
  <li>Measure fluorescence of all samples in the plate reader (settings listed above in Plate Reader Setup menu).</li>
−
<li>Record data in Fluorescein Standard Curve excel sheet and graph.</li>
+
  <li>Record data in Fluorescein Standard Curve excel sheet and graph.</li>
 
</ol>
 
</ol>
 +
 
<h1>Cell Measurements</h1>
 
<h1>Cell Measurements</h1>
 +
 
<p><strong>Materials:</strong></p>
 
<p><strong>Materials:</strong></p>
−
<ul>Competent Cells (Escherichia coli strain DH5 alpha)</ul>
 
−
<ul>Luria Bertani media</ul>
 
 
<ul>
 
<ul>
−
<ul>Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH, working stock 25 ug/mL)
+
  <li> Competent Cells (Escherichia coli strain DH5 alpha)</li>
−
<ul>50 mL Falcon tube (covered in foil to block light)</ul>
+
  <li>Luria Bertani media</li>
−
<ul>Incubator at 37 degree Celsius</ul>
+
  <li>Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH, working stock 25 ug/mL) </li>
−
<ul>1.5 mL eppendorf tubes for sample storage</ul>
+
  <li>50 mL Falcon tube (covered in foil to block light)</li>
−
<ul>Ice bucket with ice</ul>
+
  <li>Incubator at 37 degree Celsius</li>
−
<ul>Pipettes</ul>
+
  <li>1.5 mL eppendorf tubes for sample storage</li>
−
<ul>96 well plate, black with transparent bottom (Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)</ul>
+
  <li>Ice bucket with ice</li>
−
</ul>
+
  <li>Pipettes</li>
 +
  <li>96 well plate, black with transparent bottom (Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)</li>
 
</ul>
 
</ul>
 +
 
<p><br /><strong>Plasmids</strong></p>
 
<p><br /><strong>Plasmids</strong></p>
 
<ul>
 
<ul>
−
<ul>
+
  <li>BBA_J364000 (1), J23101+I13504</li>
−
<ul>
+
  <li>BBa_J364001 (2), J23106+I13504</li>
−
<li>BBA_J364000 (1), J23101+I13504</li>
+
  <li>BBa_J364002 (3), J23107+I13504</li>
−
<li>BBa_J364001 (2), J23106+I13504</li>
+
  <li>BBa_J36003 (4), J23101.BCD2.E0040.B0015</li>
−
<li>BBa_J364002 (3), J23107+I13504</li>
+
  <li>BBa_J364004 (5), J23106.BCD2.E0040.B0015</li>
−
<li>BBa_J36003 (4), J23101.BCD2.E0040.B0015</li>
+
  <li>BBa_J364005 (6), J23107.BCD2.E0040.B0015</li>
−
<li>BBa_J364004 (5), J23106.BCD2.E0040.B0015</li>
+
  <li>Positive control BBa_120270</li>
−
<li>BBa_J364005 (6), J23107.BCD2.E0040.B0015</li>
+
  <li>Negative control BBa_R0040</li>
−
<li>Positive control BBa_120270</li>
+
−
<li>Negative control BBa_R0040</li>
+
−
</ul>
+
−
</ul>
+
 
</ul>
 
</ul>
 +
 +
 
<p><br /><strong>Methods:</strong></p>
 
<p><br /><strong>Methods:</strong></p>
−
<ul>
 
 
<ol>
 
<ol>
−
<li>Transform Esherichia coli DH5 alpha with following the following plasmids
+
    <li>Transform Esherichia coli DH5 alpha with following the following plasmids</li>
−
<ul>
+
    <ul>
−
<li>Device 1: BBA_J364000 (1), J23101+I13504</li>
+
          <li>Device 1: BBA_J364000 (1), J23101+I13504</li>
−
<li>Device 2: BBa_J364001 (2), J23106+I13504</li>
+
          <li>Device 2: BBa_J364001 (2), J23106+I13504</li>
−
<li>Device 3: BBa_J364002 (3), J23107+I13504</li>
+
          <li>Device 3: BBa_J364002 (3), J23107+I13504</li>
−
<li>Device 4: BBa_J36003 (4), J23101.BCD2.E0040.B0015</li>
+
          <li>Device 4: BBa_J36003 (4), J23101.BCD2.E0040.B0015</li>
−
<li>Device 5: BBa_J364004 (5), J23106.BCD2.E0040.B0015</li>
+
          <li>Device 5: BBa_J364004 (5), J23106.BCD2.E0040.B0015</li>
−
<li>Device 6: BBa_J364005 (6), J23107.BCD2.E0040.B0015</li>
+
          <li>Device 6: BBa_J364005 (6), J23107.BCD2.E0040.B0015</li>
−
<li>Positive control BBa_120270</li>
+
          <li>Positive control BBa_120270</li>
−
<li>Negative control BBa_R0040.</li>
+
          <li>Negative control BBa_R0040.</li>
−
</ul>
+
    </ul>
−
</li>
+
   
−
<li>Pick 2 colonies from each plate and inoculate on a 5-10 mL LB medium + Chloramphenicol.
+
    <li>Pick 2 colonies from each plate and inoculate on a 5-10 mL LB medium + Chloramphenicol.</li>
−
<ul>18 tubes of 5 mL LB with 5 uL of chloramphenicol (25 ug/mL)</ul>
+
          <ul><li>18 tubes of 5 mL LB with 5 uL of chloramphenicol (25 ug/mL)</li></ul>
−
</li>
+
   
−
<li>Grow cells overnight for 16-18 hours at 37 degrees Celsius and at 220 rpm.</li>
+
    <li>Grow cells overnight for 16-18 hours at 37 degrees Celsius and at 220 rpm.</li>
−
<li>Measure cell growth with spectrophotometer:
+
    <li>Measure cell growth with spectrophotometer:</li>
−
<ul>
+
    <ul>
−
<li>Set spectrophotometer to read OD600.</li>
+
          <li>Set spectrophotometer to read OD600.</li>
−
<li>Measure OD600 of overnight cultures (placed in 1 mL spectrophotometer cuvettes) after calibrating instrument with distilled H2O.</li>
+
          <li>Measure OD600 of overnight cultures (placed in 1 mL spectrophotometer cuvettes) after calibrating instrument with distilled H2O.</li>
−
<li>Record data and transfer data into the Dilution Calculation Sheet_1 provided by iGEM Headquarters.</li>
+
          <li>Record data and transfer data into the Dilution Calculation Sheet_1 provided by iGEM Headquarters.</li>
−
<li>Dilute all cultures to a target OD600 of 0.02 in 12 mL LB medium + Chloramphenicol in a 50 mL capacity Falcon Tube covered with aluminum foil to block light.</li>
+
          <li>Dilute all cultures to a target OD600 of 0.02 in 12 mL LB medium + Chloramphenicol in a 50 mL capacity Falcon Tube covered with aluminum foil to block light.</li>
−
<li>Incubate cultures at 37 degrees Celsius and at 220 rpm.</li>
+
          <li>Incubate cultures at 37 degrees Celsius and at 220 rpm.</li>
−
<li>Take 500 uL samples of each of the cultures at 0 hours, 2 hours, 4 hours and 6 hours of incubation.</li>
+
          <li>Take 500 uL samples of each of the cultures at 0 hours, 2 hours, 4 hours and 6 hours of incubation.</li>
−
<li>Total samples taken should be 16 samples, with 2 samples from each 8 devices at each time point.</li>
+
          <li>Total samples taken should be 16 samples, with 2 samples from each 8 devices at each time point.</li>
−
<li>Do not reuse samples</li>
+
          <li>Do not reuse samples</li>
−
</ul>
+
    </ul>
−
</li>
+
    <li>Place samples on ice in meantime while pipetting them into the plate to be measured.</li>
−
<li>Place samples on ice in meantime while pipetting them into the plate to be measured.
+
    <ul>
−
<ul>
+
          <li>When pipetting into the clear bottom plate to be measured by the TECAN Infinite M1000 plate reader using absorbance and fluorescence:</li>
−
<li>When pipetting into the clear bottom plate to be measured by the TECAN Infinite M1000 plate reader using absorbance and fluorescence:
+
          <ol>
−
<ol>
+
                <li>Pipette both samples into replicates of 4 each so that there are 4 wells, each filled with 100 uL, for each sample.</li>
−
<li>Pipette both samples into replicates of 4 each so that there are 4 wells, each filled with 100 uL, for each sample.</li>
+
                <li>This totals to 8 wells of 100 uL per device.</li>
−
<li>This totals to 8 wells of 100 uL per device.</li>
+
          </ol>
 +
      </ul>
 +
      <li>Plate setup should look like</li>
 +
      <img src="https://static.igem.org/mediawiki/2017/4/4d/Team_McMasterU_Plate_Setup.jpg" alt="Plate Setup" /></li>
 +
      <li>Measure with plate reader with both absorbance and fluorescence.</li>
 +
      <li>Record data into cell measurement tab of the Excel Sheet_1 provided.</li>
 
</ol>
 
</ol>
−
</li>
+
 
−
</ul>
+
−
</li>
+
−
<li>Plate setup should look like</li>
+
−
<li><img src="http://paste.pics/277OM" alt="Plate Setup" /></li>
+
−
<li>Measure with plate reader with both absorbance and fluorescence.</li>
+
−
<li>Record data into cell measurement tab of the Excel Sheet_1 provided.</li>
+
−
<li><br /><br />
+
 
<h1>Results</h1>
 
<h1>Results</h1>
 
<br />
 
<br />
 +
 
<h2>OD600 Calibration</h2>
 
<h2>OD600 Calibration</h2>
−
<img src="http://paste.pics/277QE" alt="OD600 Calibration" /> <br /> <img src="http://paste.pics/277QJ" alt="OD600 of LUDOX 100% and dH2O" /> <br />
+
<img src="https://static.igem.org/mediawiki/2017/0/0b/Team_McMasterU_OD600_Calibration.jpg" alt="OD600 Calibration" /> <br /> <img src="https://static.igem.org/mediawiki/2017/d/d2/Team_McMasterU_OD600of_LUDOX.jpg" alt="OD600 of LUDOX 100% and dH2O" /> <br />
 
<h2>Fluorescein Standard Curve</h2>
 
<h2>Fluorescein Standard Curve</h2>
−
<img src="http://paste.pics/277QU" alt="Fluorescein Standard Curve Table" /> <br /> <img src="http://paste.pics/277R2" alt="Fluorescein Standard Curve" /> <br /> <img src="http://paste.pics/277R7" alt="Fluorescein Standard Curve (log curve)" /> <br /> <br />
+
<img src="https://static.igem.org/mediawiki/2017/1/10/Team_McMasterU_Fluorescein_Standard_Curve_Table.jpg" alt="Fluorescein Standard Curve Table" /> <br /> <img src="https://static.igem.org/mediawiki/2017/9/90/Team_McMasterU_Fluorescein_Standard_Curve.jpg" alt="Fluorescein Standard Curve" /> <br /> <img src="https://static.igem.org/mediawiki/2017/e/eb/Team_McMasterU_Fluorescein_Standard_Curve_%28log_curve%29.jpg" alt="Fluorescein Standard Curve (log curve)" /> <br /> <br />
 +
 
 
<h2>Dilution Calculations</h2>
 
<h2>Dilution Calculations</h2>
−
<img src="http://paste.pics/277RF" alt="Dilution Calculations Table" /> <br /> <br />
+
<img src="https://static.igem.org/mediawiki/2017/6/6c/Team_McMasterU_Dilution_Calculations_Table.jpg" alt="Dilution Calculations Table" /> <br /> <br />
 +
 
 
<h2>OD Readings</h2>
 
<h2>OD Readings</h2>
−
<img src="http://paste.pics/277RR" alt="Average OD600 Absorbance Value of Devices Measured over 6 hours" /> <img src="http://paste.pics/277RW" alt="Table of Average OD600 Values" /> <br /> <br />
+
<img src="https://static.igem.org/mediawiki/2017/9/90/Team_McMasterU_Average_OD600_Absorbance_Value_of_Devices_Measured_over_6_hours.jpg" alt="Average OD600 Absorbance Value of Devices Measured over 6 hours" /> <img src="https://static.igem.org/mediawiki/2017/3/3e/Team_McMasterU_Table_of_Average_OD600_Values.jpg" alt="Table of Average OD600 Values" /> <br /> <br />
 +
 
 
<h2>Fluorescence Readings</h2>
 
<h2>Fluorescence Readings</h2>
−
<img src="http://paste.pics/277S2" alt="Fluorescence Averages of Devices over 6 Hours" /> <br /> <img src="http://paste.pics/277S7" alt="Fluorescence Averages of Devices over 6 Hours" /> <br />
+
<img src="https://static.igem.org/mediawiki/2017/d/d7/Team_McMasterU_Fluorescence_Averages_of_Devices_over_6_Hours.jpg" alt="Fluorescence Averages of Devices over 6 Hours" /> <br /> <img src="https://static.igem.org/mediawiki/2017/8/8b/Team_McMasterU_Fluorescence_Averages_of_Devices_over_6_Hours2.jpg" alt="Fluorescence Averages of Devices over 6 Hours" /> <br />
 +
 
  
−
</li>
 
−
</ol>
 
−
</ul>
 
  
 
</div>
 
</div>
 
</html>
 
</html>
 
{{:Template:McMasterU_Footer}}
 
{{:Template:McMasterU_Footer}}

Latest revision as of 19:21, 29 October 2017

Overview

The Interlab Study, developed by the Measurement Committee of iGEM as a way to standardize measurements for green fluorescent protein (GFP), tackles the foundational synthetic biology problem of a lack of reliable measurements for experiments. With an international standard set down, scientists can go beyond utilizing relativity comparisons for results, to conduct quantitative reproducibility studies across labs. This Interlab experiment, carried out by McMaster iGEM with plate readers, aims to produce comparable GFP measurements as a contribution for the international standardization of GFP values. The parts we used were BBA_J364000 (1), BBa_J364001 (2), BBa_J364002 (3), BBa_J36003 (4), BBa_J364004 (5), BBa_J364005 (6), and positive control BBa_120270 with negative control BBa_R0040. All parts were located in pSB1C3 plasmid and were modified for chloramphenicol resistance.

Methods and Materials

Plate Reader

Plate Reader: TECAN Infinite M1000 Plate: Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)
Abs OD600

  • Wavelengths: 600 nm
  • Number of Flashes: 20
  • Settle Time: 0 ms
  • Temperature: 26.1 degrees Celsius

Fluorescence

  • Excitation:485 nm
  • Emission: 535 nm
  • Excitation Bandwidth: 5 nm
  • Emission Bandwidth: 5 nm
  • Optics: Bottom Reading
  • Gain: Optimal (automatic)
  • Number of Flashes: 50
  • Flash Frequency: 400 Hz
  • Integration Time: 20 us
  • Lag Time: 0 us
  • Settle Time: 0 ms
  • Part of Plate: A1-H10
  • Temperature: 26.8 degrees Celsius



OD600 Reference Point

Materials

  • 1 mL LUDOX (provided for by iGEM Headquarters)
  • Distilled H2O
  • 96 well plate, Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)


Methods

  1. Add 100 uL of LUDOX into A1, B1, C1, and D1 wells
  2. Add 1 mL LUDOX into a separate cuvette
  3. Add 100 uL of dH2O into wells A2, B2, C2, D2 wells
  4. Add 1 mL dH2O into a separate cuvette
  5. Image using plate reader (TECAN Infinite M1000)



Protocol of Fluorescein Fluorescence Standard Curve

Materials:

  • Fluorescein
  • 10 mL 1x PBS (phosphate buffered saline)
  • 96 well plate, Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)


Methods:

  1. Spin down fluorescence stock tube until pellet is at bottom of tube
  2. Resuspend 100 uM stock Fluorescein in 1 mL of 1x PBS to obtain 2x fluorescein stock solution.
  3. Ensure that Fluorescein is properly dissolved.
  4. Dilute 2x fluorescin stock solution with 1x PBS to make a 1x fluorescein solution. Resulting concentration of fluorescein stock solution should be 50 uM (otherwise known as 500 uL of 2x fluoresceine in 500 uL of 1x PBS that makes 1 mL of 50 uM of 1x fluorescein solution).
  5. Prepare for serial dilutions across columns 1 to 11. Ensure column 12 has PBS buffer only.
    • Pipette 200 uL of 1x fluorescein stock into wells A1, B1, C1, D1.
    • Pipette 100 uL of PBS in wells A2, B2, C2, D2 ... A12, B12.
    • Transfer 100 uL of fluorescein stock solution from A1 to A2.
    • Mix A2 by pipetting up and down 3x and transfer 100 uL into A3.
    • Mix A3 by pipetting up and down 3x and transfer 100 uL into A4.
    • Repeat 5c to 5e for A4, A5, A6.... to A11.
    • Mix A11 by pipetting up and down 3x and then transfer 100 uL into liquid waste. Ensure that A12 is PBS only
  6. Repeat step 5 for columns B, C, and D.
  7. Measure fluorescence of all samples in the plate reader (settings listed above in Plate Reader Setup menu).
  8. Record data in Fluorescein Standard Curve excel sheet and graph.

Cell Measurements

Materials:

  • Competent Cells (Escherichia coli strain DH5 alpha)
  • Luria Bertani media
  • Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH, working stock 25 ug/mL)
  • 50 mL Falcon tube (covered in foil to block light)
  • Incubator at 37 degree Celsius
  • 1.5 mL eppendorf tubes for sample storage
  • Ice bucket with ice
  • Pipettes
  • 96 well plate, black with transparent bottom (Corning 96 Flat Bottom black, clear bottom Polystyrol (COR96fb clear bottom)


Plasmids

  • BBA_J364000 (1), J23101+I13504
  • BBa_J364001 (2), J23106+I13504
  • BBa_J364002 (3), J23107+I13504
  • BBa_J36003 (4), J23101.BCD2.E0040.B0015
  • BBa_J364004 (5), J23106.BCD2.E0040.B0015
  • BBa_J364005 (6), J23107.BCD2.E0040.B0015
  • Positive control BBa_120270
  • Negative control BBa_R0040


Methods:

  1. Transform Esherichia coli DH5 alpha with following the following plasmids
    • Device 1: BBA_J364000 (1), J23101+I13504
    • Device 2: BBa_J364001 (2), J23106+I13504
    • Device 3: BBa_J364002 (3), J23107+I13504
    • Device 4: BBa_J36003 (4), J23101.BCD2.E0040.B0015
    • Device 5: BBa_J364004 (5), J23106.BCD2.E0040.B0015
    • Device 6: BBa_J364005 (6), J23107.BCD2.E0040.B0015
    • Positive control BBa_120270
    • Negative control BBa_R0040.
  2. Pick 2 colonies from each plate and inoculate on a 5-10 mL LB medium + Chloramphenicol.
    • 18 tubes of 5 mL LB with 5 uL of chloramphenicol (25 ug/mL)
  3. Grow cells overnight for 16-18 hours at 37 degrees Celsius and at 220 rpm.
  4. Measure cell growth with spectrophotometer:
    • Set spectrophotometer to read OD600.
    • Measure OD600 of overnight cultures (placed in 1 mL spectrophotometer cuvettes) after calibrating instrument with distilled H2O.
    • Record data and transfer data into the Dilution Calculation Sheet_1 provided by iGEM Headquarters.
    • Dilute all cultures to a target OD600 of 0.02 in 12 mL LB medium + Chloramphenicol in a 50 mL capacity Falcon Tube covered with aluminum foil to block light.
    • Incubate cultures at 37 degrees Celsius and at 220 rpm.
    • Take 500 uL samples of each of the cultures at 0 hours, 2 hours, 4 hours and 6 hours of incubation.
    • Total samples taken should be 16 samples, with 2 samples from each 8 devices at each time point.
    • Do not reuse samples
  5. Place samples on ice in meantime while pipetting them into the plate to be measured.
    • When pipetting into the clear bottom plate to be measured by the TECAN Infinite M1000 plate reader using absorbance and fluorescence:
      1. Pipette both samples into replicates of 4 each so that there are 4 wells, each filled with 100 uL, for each sample.
      2. This totals to 8 wells of 100 uL per device.
  6. Plate setup should look like
    7. Plate Setup
  7. Measure with plate reader with both absorbance and fluorescence.
  8. Record data into cell measurement tab of the Excel Sheet_1 provided.

Results


OD600 Calibration

OD600 Calibration
OD600 of LUDOX 100% and dH2O

Fluorescein Standard Curve

Fluorescein Standard Curve Table
Fluorescein Standard Curve
Fluorescein Standard Curve (log curve)

Dilution Calculations

Dilution Calculations Table

OD Readings

Average OD600 Absorbance Value of Devices Measured over 6 hours Table of Average OD600 Values

Fluorescence Readings

Fluorescence Averages of Devices over 6 Hours
Fluorescence Averages of Devices over 6 Hours