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+ | <h1>INTERLAB</h1> | ||
+ | <u><b>Calibration 1: OD600 Reference point</b></u> | ||
+ | <ul> | ||
+ | <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> | ||
+ | <br /> | ||
+ | <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>Results: Cell density readings can thus be converted to OD<sub>600</sub> by multiplying correction factor value, <b>4.138</b>.</li> | ||
+ | </ul> | ||
+ | <br /> | ||
− | < | + | <i>Table1.</i> |
− | + | <p>Table shows absorbance measurements (at 600 nm) for LUDOX CL-X and dd-H2O using a plate reader. The corrected Abs<sub>600</sub> is the difference between the LUDOX CL-X reading and dd H2O reading. Reference OD<sub>600</sub> is a measurement by a spectrophotometer (provided on iGEM excel sheet). OD<sub>600</sub>/Abs<sub>600</sub> is the correction factor to convert Abs<sub>600</sub> to OD<sub>600</sub>, calculated by dividing Reference OD<sub>600</sub> by Abs<sub>600</sub>.</p> | |
− | <p> | + | <img src="https://static.igem.org/mediawiki/2018/d/d0/T--Toronto--7_25_2018_Interlab_LUDOX_CL_X.png" alt="LUDOX_CL_X"> |
− | < | + | <br /> |
− | </ | + | <br /> |
+ | <u><b>Calibration 2: Particle Standard Curve</u></b> | ||
+ | <p>Construct a standard curve of Abs<sub>600</sub> 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. | ||
+ | </p> | ||
+ | <ul> | ||
+ | <li>Use standard curve to convert Abs<sub>600</sub> measurements to an estimate of number of cells.</li> | ||
+ | <li>Monodisperse silica microspheres are used in the calibration because they have similar size and optical characteristics as cells.</li> | ||
+ | </ul> | ||
− | < | + | (a) |
+ | <img src="https://static.igem.org/mediawiki/2018/3/3e/T--Toronto--7_25_2018_Interlab_graph.png" alt="graph"> | ||
+ | |||
+ | (b) | ||
+ | <img src="https://static.igem.org/mediawiki/2018/b/b1/T--Toronto--7_25_2018_Interlab_graph1.png" alt="graph1"> | ||
+ | <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/100μL 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 Abs<sub>600</sub> 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 (Abs<sub>600</sub> 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> | ||
+ | <br /> | ||
− | + | <u><b>Colony Forming Units per 0.1 OD<sub>600</sub> E. coli cultures</b></u> | |
− | + | <p>Purpose of this calibration: Determine a conversion method that can be used to estimate cell concentration per mL by using Colony Forming Unit (CFU) counts while assuming that every colony arises from a singular bacterial cell.</p> | |
− | + | ||
− | 0. | + | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
+ | <ul> | ||
+ | <li>1:100 dilution of overnight cultures of negative and positive controls.</li> | ||
+ | <li>Calibration of OD<sub>600</sub> to CFU counts.</li> | ||
+ | <li> | ||
+ | Counted colonies for the two Positive Control (BBa_I20270) and Negative Control (BBa_R0040) cultures. | ||
+ | <ul> | ||
+ | <li>Starting Sample Preparation was conducted to achieve OD<sub>600</sub> of 0.1 for the overnight cultures.</li> | ||
+ | <li>Serial Dilutions conducted, colony count conducted for the final dilution factors of 8x10^4, 8x10^5, and 8x10^6.</li> | ||
+ | </ul> | ||
+ | </li></ul> | ||
+ | <br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/a/a8/T--Toronto--7_25_2018_Interlab_picture2.png" alt="picture2"> | ||
+ | <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 /> | ||
− | + | <i>Table 2.:</i> | |
− | + | <p>CFU Colony count for the two colonies of positive and negative control (2B and 2D respectively).</p> | |
+ | <img src="https://static.igem.org/mediawiki/2018/c/c9/T--Toronto--7_25_2018_Interlab_table2.png" alt="table2"> | ||
+ | <br /> | ||
+ | <br /> | ||
+ | <i>Table 3.:</i> | ||
+ | <u>Colony forming unit calculation (using Dilution 3 data)</u> | ||
+ | <p>(# of colonies) x (Final Dilution Factor) = <b>CFU/mL</b></p> | ||
+ | <p>Final Dilution Factor = 8 x 10^4.</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/a/aa/T--Toronto--7_25_2018_Interlab_table3.png" alt="table3"> | ||
+ | <p>Conclusion: For a starting culture of Colony 1 of Positive Control (2B) cells at OD<sub>600</sub> = 0.1 when dilution factor is 8x10^4, there is an average of 7.62x10^6 CFUs/mL.</p> | ||
+ | <br /> | ||
− | + | <h3>Appendix</h3> | |
− | + | <b>Raw Plate Readings</b> | |
− | </ | + | |
+ | <b>Assumed well plate pattern for the following tables:</b> | ||
+ | <br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/b/b7/T--Toronto--7_25_2018_Interlab_Capture.png" alt="Capture"> | ||
+ | <br /> | ||
+ | <br /> | ||
− | < | + | <b>Fluorescence Raw Readings:</b> |
− | < | + | <br /> |
− | <br | + | <img src="https://static.igem.org/mediawiki/2018/3/37/T--Toronto--7_25_2018_Interlab_Capture1.png" alt="Capture1"> |
− | + | <br /> | |
+ | <img src="https://static.igem.org/mediawiki/2018/a/a5/T--Toronto--7_25_2018_Interlab_Capture2.png" alt="Capture2"> | ||
+ | <br /> | ||
+ | <br /> | ||
− | </ | + | <b>Abs<sub>600</sub> Raw Readings:</b> |
− | </ | + | <br /> |
+ | <img src="https://static.igem.org/mediawiki/2018/7/7f/T--Toronto--7_25_2018_Interlab_Capture3.png" alt="Capture3"> | ||
+ | <br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/a/ae/T--Toronto--7_25_2018_Interlab_Capture4.png" alt="Capture4"> | ||
+ | </div> | ||
+ | </body> | ||
− | </html> | + | </html> |
Latest revision as of 13:52, 30 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:
- 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.
![lambert-law](https://static.igem.org/mediawiki/2018/b/b5/T--Toronto--7_25_2018_Interlab_lambert-law.gif)
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](https://static.igem.org/mediawiki/2018/d/d0/T--Toronto--7_25_2018_Interlab_LUDOX_CL_X.png)
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](https://static.igem.org/mediawiki/2018/3/3e/T--Toronto--7_25_2018_Interlab_graph.png)
![graph1](https://static.igem.org/mediawiki/2018/b/b1/T--Toronto--7_25_2018_Interlab_graph1.png)
Figure 1. Calibration of particle count to absorbance measured by plate-reader (a) A particle standard curve of Abs600 for known particle count/100μL 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.
![graph2](https://static.igem.org/mediawiki/2018/8/80/T--Toronto--7_25_2018_Interlab_graph2.png)
![graph3](https://static.igem.org/mediawiki/2018/b/bb/T--Toronto--7_25_2018_Interlab_graph3.png)
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](https://static.igem.org/mediawiki/2018/7/7c/T--Toronto--7_25_2018_Interlab_picture.png)
“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](https://static.igem.org/mediawiki/2018/9/9c/T--Toronto--7_25_2018_Interlab_picture1.png)
“IGEM 2018 InterLab Study Protocol.” IGEM 2018, IGEM Foundation, 2018, 2018.igem.org/wiki/images/0/09/2018_InterLab_Plate_Reader_Protocol.pdf.
Colony Forming Units per 0.1 OD600 E. coli cultures
Purpose of this calibration: Determine a conversion method that can be used to estimate cell concentration per mL by using Colony Forming Unit (CFU) counts while assuming that every colony arises from a singular bacterial cell.
- 1:100 dilution of overnight cultures of negative and positive controls.
- Calibration of OD600 to CFU counts.
-
Counted colonies for the two Positive Control (BBa_I20270) and Negative Control (BBa_R0040) cultures.
- Starting Sample Preparation was conducted to achieve OD600 of 0.1 for the overnight cultures.
- Serial Dilutions conducted, colony count conducted for the final dilution factors of 8x10^4, 8x10^5, and 8x10^6.
![picture2](https://static.igem.org/mediawiki/2018/a/a8/T--Toronto--7_25_2018_Interlab_picture2.png)
“IGEM 2018 InterLab Study Protocol.” IGEM 2018, IGEM Foundation, 2018, 2018.igem.org/wiki/images/0/09/2018_InterLab_Plate_Reader_Protocol.pdf.
Table 2.:
CFU Colony count for the two colonies of positive and negative control (2B and 2D respectively).
![table2](https://static.igem.org/mediawiki/2018/c/c9/T--Toronto--7_25_2018_Interlab_table2.png)
Table 3.: Colony forming unit calculation (using Dilution 3 data)
(# of colonies) x (Final Dilution Factor) = CFU/mL
Final Dilution Factor = 8 x 10^4.
![table3](https://static.igem.org/mediawiki/2018/a/aa/T--Toronto--7_25_2018_Interlab_table3.png)
Conclusion: For a starting culture of Colony 1 of Positive Control (2B) cells at OD600 = 0.1 when dilution factor is 8x10^4, there is an average of 7.62x10^6 CFUs/mL.
Appendix
Raw Plate Readings Assumed well plate pattern for the following tables:![Capture](https://static.igem.org/mediawiki/2018/b/b7/T--Toronto--7_25_2018_Interlab_Capture.png)
Fluorescence Raw Readings:
![Capture1](https://static.igem.org/mediawiki/2018/3/37/T--Toronto--7_25_2018_Interlab_Capture1.png)
![Capture2](https://static.igem.org/mediawiki/2018/a/a5/T--Toronto--7_25_2018_Interlab_Capture2.png)
Abs600 Raw Readings:
![Capture3](https://static.igem.org/mediawiki/2018/7/7f/T--Toronto--7_25_2018_Interlab_Capture3.png)
![Capture4](https://static.igem.org/mediawiki/2018/a/ae/T--Toronto--7_25_2018_Interlab_Capture4.png)