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| − | <p>INTERLAB STUDY | + | <p> |
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| − | Our iGEM Paris Bettencourt 2018 team wanted to take part of the Interlab study.
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| − | The aim is to improve the measurement tools for synthetic biology : allowing reliable and reproducible measurements.
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| − | To do so, teams world-wide participated in a global experiment to follow the same measurement experiment protocol and share their results, this way, we could compare and understand the source of variable results.
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| − | This year we wanted to know if we can reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?
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| − | PROTOCOL :
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| − | First of all, every team should do calibrations using LUDOX CL-X, silica beads and fluorescein.
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| − | CALIBRATION 1 WITH LUDOX CL-X :
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| − | LUDOX CL-X is used as a reference to transform the plate reader absorbance data into a comparable spectrophotometer OD600 measurement.
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| − | We prepared 4 replicates of 100 µL LUDOX CL-X samples and 100 µL ddH2O samples, measured absorbance at 600 nm with our TECAN plate reader and record data.
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| − | CALIBRATION 2 WITH SILICA BEADS :
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| − | The silica beads provided by iGEM have the same size and shape as Escherichia coli cells, thus, it can scatter light the same way. We measure absorbance to approximate the concentration of cells in the sample according to the way the cells scatter light in a liquid.
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| − | We did a serial dilution of the silica beads sample in ddH2O, then, we measured absorbance in our plate reader and record data.
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| − | CALIBRATION 3 WITH FLUORESCEIN :
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| − | Each instrument reports fluorescence in arbitrary units so that it is difficult to compare values from different instruments .The aim is to create a standard fluorescence curve for all the teams. Fluorescein has the same excitation and emission properties as GFP.
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| − | We prepared a dilutions series of fluorescein in four replicates and measure the fluorescence in a 96 well plate reader. With the data, we can generate a standard curve of fluorescence which we can use to convert our cell based reading to and equivalent fluorescein concentration.
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| − | After these calibrations experiments, we could prepare competent cells, measure their efficiency and start the cell measurement protocol.
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| − | CELL MEASUREMENT PROTOCOL :
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| − | We had to transform Escherichia coli DH5 a with 8 differents plasmids. Then, pick colonies from each transformation, inoculate them in LB medium + chloramphenicol and grow them overnight. We did 4 replicates for each colony. After dilution, we measured fluorescence before and after 6 hours of incubation and recorded the data.
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| − | COUNTING CFU PER 0.1 OD600 ESCHERICHIA COLI CULTURES :
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| − | The aim of this protocol to calibrate OD600 to colony forming unit (CFU) counts. These are relatable to the cell concentration of the culture.
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| − | We did serial dilutions for triplicates of the previous positive and negative controls, measured OD600 of our cell cultures and dilute them until an 0.1 OD measurement.
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| − | Then, we repeated the same serial dilutions for our triplicates samples, plated them and incubated them overnight. We counted colonies after this.
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| − | Based on the assumption that 1 bacterial cell gives rise to 1 colony, we could calculate CFU per 1mL of our 0.1 OD600 culture.
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| | </p> | | </p> |
paris-bettencourt-2018@cri-paris.org 
