Difference between revisions of "Team:Newcastle/InterLab"

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        Calibrations
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        Protocol
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                            <h3 class="subhead">Newcastle InterLab Study</h3>
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                 <h1 class="display-1">Overview</h1>
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                 <h1 class="display-2">Calibration</h1>
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                    <p style="font-size:100%">Reproducibility, the ability to carry out and reproduce the results of a single experiment, is an important aspect of scientific disciplines. However, in the life sciences, the concept of reproducibility has become a large problem. A vast number of experiments throughout the various disciplines of the life sciences are seen to lack a reproducible nature, ultimately costing and inadvertently wasting large sums of money. Synthetic biology is no exception to the troubles of irreproducibility, with inaccurate part characterisation impacting the the ability to use Bio-Design Automation (BDA) to build fully functioning, novel synthetic gene circuits.</p>
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                <p><font size="3">Alternative Roots participated in the 2018 InterLab study. Three calibration steps were carried out prior to any experimental measurements being taken. Absorbance and fluorescence values were measured in 96-well plates using a Thermofisher Varioskan Lux plate reader (Thermofisher Scientific). Absorbance was measured at 600 nm and converted to a comparable OD<sub>600</sub>. Fluorescence was measured at 525 nm with excitation at 485 nm with a 12 nm bandpass width. All readings took place at 25 °C and pathlength correction was disabled. The calibrations were:</p>
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                    <p style="font-size:100%">iGEM devised the inter-lab study – an annual, large-scale study carried out by institutions around the world by researchers of varying experience levels – to determine the reproducibility of individual synthetic biology protocols and to assess and address the limiting factors of reproducibility. The study attempted to address variation between different models of plate readers by producing a step by step protocol for measurement and analysis, allowing the production of directly comparable fluorescence measurements. In addition, ribosome binding site (RBS) sequences designed to increase precision of expression were included in the devices to be transformed into the host cells.</p>
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                    <p style="font-size:100%">A weakness in the measurement of fluorescence relative to OD600, as with previous IGEM interlab protocols, is the potential discrepancy between optical density and actual cell concentration. This year the IGEM study aims to reduce lab-to-lab variability further by measuring GFP fluorescence relative to absolute cell counts or colony forming units. Normalisation of fluorescence to colony forming units goes further by allowing measurement of fluorescence relative only to viable cells, and thus a more accurate measurement of promoter strength, whereas OD600 and absolute cell count measures cannot differentiate between viable and non-viable cells.</p>
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<p><font size="3">1. A LUDOX CL-X 45 % colloidal silica suspension was used to calculate a conversion factor for the Abs<sub>600</sub> value measured by the plate reader to a comparable OD<sub>600</sub> value, considering path length and well volume (Table 1). Abs<sub>600</sub> of 1:2 dilutions of LUDOX silica suspension were taken in triplicate and a reference OD<sub>600</sub> of 0.063 (the reference value for 100 µL of LUDOX CL-X in a well of a standard 96-well flat-bottom black with clear bottom plate) divided by the mean measured value to give a conversion factor. </font></p>
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<p><font size="2"><center>Table 1. Optical density readings for LUDOX CL-X 45% colloidal silica suspension and water used to calculate the conversion factor for absorbance readings to OD<sub>600</sub> readings for plate reader measurements.
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<p><font size="3">2. A standard curve was prepared by measuring the OD<sub>600</sub> of serial dilutions of monodisperse silica microspheres, with similar light scattering properties to <i>E. coli</i> cells. This was used to standardise OD readings across labs (Figure 1A). </p>
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<p><font size="3">3. A fluorescence standard curve was created by measuring the fluorescence of serial dilutions of the small molecule fluorescein. This  has similar excitation and emission characteristics to GFP allowing conversion of fluorescence readings to an equivalent fluorescein concentration. Calibrations allowed expression measurement in units of fluorescence per OD and molecules of equivalent fluorescein (MEFL) per cell (Figure 1B). </p>
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<p><font size="2"><center>Figure 1. Curves used to calibrate A; Fluorescein per OD using dilutions of fluorescein and B; molecules of equivalent fluorescein per particle using dilutions of solutions of monodisperse silica microspheres.</font></center></p>
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                <p><font size="3">Following these calibration steps, two transformed colonies for each test device and both control plasmids were used to inoculate lysogeny broth (LB) medium containing 25 µg/L of chloramphenicol (CAM) and incubated overnight at 37 °C with shaking at 220 rpm. Overnight cultures were diluted 1:10 and the OD<sub>600</sub> adjusted to 0.02 with LB with CAM to a final volume of 12 mL. Fluorescence and Abs<sub>600</sub> were taken at 0h and 6 hours of incubation at 37 °C with 220 rpm shaking. Test devices and plasmid backbone are shown in Figure 2.</font></p>
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                    <p style="text-align:center"><br>The engineers, hard at work trying to troubleshoot issues with the system.</p>
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<p><font size="2"><center>Figure 2. A) test devices transform into DH5α for the iGEM InterLab study. A negative control device consisting of a tetracycline resistance gene (BBa_R0040) with no associated promoter, RBS or terminator sequences was also transformed into DH5α. B) All test devices were transformed into DH5α using the pSB1C3 plasmid backbone with chloramphenicol for selection (adapted from LabGenius plasmid viewer).</font></center></p>
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                    <p style="text-align:center"><br>The finished product, set to a rainbow function that cycles through various wavelengths of light</p>
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                <p><font size="3">Results for OD<sub>600</sub> and fluorescence measurements after 6 hours are shown in Figure 3.</font></p>
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<p><font size="2"><center>Figure 3. OD<sub>600</sub> (A) and fluorescence (B) InterLab study results for positive and negative controls and all test devices after 6 hours. Tests were performed in duplicate using two colonies for each test device, results for which are shown separately to account for colony-to-colony variation. </center></font></p>
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<p><font size="3">Results for both 0 and 6 hours were converted into fluorescence per OD<sub>600</sub> and fluorescence per cell using the previously described calibrations (Figure 4). While the InterLab study is an opportunity for crowdsourcing a large amount of data on the inherent variability of a given biological system, it does not account for variations within data sets. Our team attempted to seek out and address such sources of variation, investigating biodesign automation of protocols, use of internal standards and effects of media composition. Further detail and results are available on the <a href="https://2018.igem.org/Team:Newcastle/Measurement"class="black">measurements page</a>.</font></p>
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<p><font size="2"><center>Figure 4. iGEM InterLab study results for positive and negative controls and all test devices. Fluorescence was measured at 525 nm and readings were taken at 0 hours and 6 hours in a 96 well plate.  A: Fluorescence per OD<sub>600</sub>, B: Fluorescence per <i>Escherichia coli</i> cell, calibrated to a standard curve of LUDOX silica beads. </center></font></p>
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                <h1 class="display-2">References & Attributions</h1>
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<p class="about-para"><strong>Attributions: Matthew Burridge, Kyle Stanforth, Sam Went
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Latest revision as of 02:04, 18 October 2018

Alternative Roots

InterLab Study

Calibrations Protocol Results

Calibration

Alternative Roots participated in the 2018 InterLab study. Three calibration steps were carried out prior to any experimental measurements being taken. Absorbance and fluorescence values were measured in 96-well plates using a Thermofisher Varioskan Lux plate reader (Thermofisher Scientific). Absorbance was measured at 600 nm and converted to a comparable OD600. Fluorescence was measured at 525 nm with excitation at 485 nm with a 12 nm bandpass width. All readings took place at 25 °C and pathlength correction was disabled. The calibrations were:

1. A LUDOX CL-X 45 % colloidal silica suspension was used to calculate a conversion factor for the Abs600 value measured by the plate reader to a comparable OD600 value, considering path length and well volume (Table 1). Abs600 of 1:2 dilutions of LUDOX silica suspension were taken in triplicate and a reference OD600 of 0.063 (the reference value for 100 µL of LUDOX CL-X in a well of a standard 96-well flat-bottom black with clear bottom plate) divided by the mean measured value to give a conversion factor.

Table 1. Optical density readings for LUDOX CL-X 45% colloidal silica suspension and water used to calculate the conversion factor for absorbance readings to OD600 readings for plate reader measurements.



2. A standard curve was prepared by measuring the OD600 of serial dilutions of monodisperse silica microspheres, with similar light scattering properties to E. coli cells. This was used to standardise OD readings across labs (Figure 1A).

3. A fluorescence standard curve was created by measuring the fluorescence of serial dilutions of the small molecule fluorescein. This has similar excitation and emission characteristics to GFP allowing conversion of fluorescence readings to an equivalent fluorescein concentration. Calibrations allowed expression measurement in units of fluorescence per OD and molecules of equivalent fluorescein (MEFL) per cell (Figure 1B).



Figure 1. Curves used to calibrate A; Fluorescein per OD using dilutions of fluorescein and B; molecules of equivalent fluorescein per particle using dilutions of solutions of monodisperse silica microspheres.

Protocol

Following these calibration steps, two transformed colonies for each test device and both control plasmids were used to inoculate lysogeny broth (LB) medium containing 25 µg/L of chloramphenicol (CAM) and incubated overnight at 37 °C with shaking at 220 rpm. Overnight cultures were diluted 1:10 and the OD600 adjusted to 0.02 with LB with CAM to a final volume of 12 mL. Fluorescence and Abs600 were taken at 0h and 6 hours of incubation at 37 °C with 220 rpm shaking. Test devices and plasmid backbone are shown in Figure 2.

Figure 2. A) test devices transform into DH5α for the iGEM InterLab study. A negative control device consisting of a tetracycline resistance gene (BBa_R0040) with no associated promoter, RBS or terminator sequences was also transformed into DH5α. B) All test devices were transformed into DH5α using the pSB1C3 plasmid backbone with chloramphenicol for selection (adapted from LabGenius plasmid viewer).

Results

Results for OD600 and fluorescence measurements after 6 hours are shown in Figure 3.

Figure 3. OD600 (A) and fluorescence (B) InterLab study results for positive and negative controls and all test devices after 6 hours. Tests were performed in duplicate using two colonies for each test device, results for which are shown separately to account for colony-to-colony variation.

Results for both 0 and 6 hours were converted into fluorescence per OD600 and fluorescence per cell using the previously described calibrations (Figure 4). While the InterLab study is an opportunity for crowdsourcing a large amount of data on the inherent variability of a given biological system, it does not account for variations within data sets. Our team attempted to seek out and address such sources of variation, investigating biodesign automation of protocols, use of internal standards and effects of media composition. Further detail and results are available on the measurements page.

Figure 4. iGEM InterLab study results for positive and negative controls and all test devices. Fluorescence was measured at 525 nm and readings were taken at 0 hours and 6 hours in a 96 well plate. A: Fluorescence per OD600, B: Fluorescence per Escherichia coli cell, calibrated to a standard curve of LUDOX silica beads.





References & Attributions

Attributions: Matthew Burridge, Kyle Stanforth, Sam Went