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    <h3>Interlab Study</h3>
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                <div class="section" id="Introduction">
                    <h2>Introduction</h2>
                    <div class="section" id="Overview">
                        <h3>Overview</h3>
                        <p>
                            One of the fundamental principles of synthetic biology is the characterization of standard biological parts. iGEM HQ's
                            International InterLab Measurement Study is an endeavour at achieving the large-scale characterization of a set of
                            biological devices which has been previously designed and catalogued in the BioBricks Registry. In this study, iGEM teams
                            from across the world set out to measure the fluorescence of model organisms which have been transformed with devices
                            comprising the same green fluorescent protein (GFP) gene expressed at different levels by being fused to promoters of
                            different strengths. This would provide a large dataset from which a more accurate analysis that accounts for lab-to-lab
                            variations can be drawn.
                        </p>
                        <p>
                            The promoters characterized in the 2015 InterLab Study are three constitutive promoters of different strengths found
                            within the <a href="http://parts.igem.org/Promoters/Catalog/Anderson">Anderson Promoter Collection</a>, which were included
                            in the 2015 iGEM Distribution Kit. We assembled each of these three promoters upstream of a given GFP-encoding BioBrick and
                            transformed each of the composite parts into three different strains of E. coli to measure their respective fluorescence levels.
                            We measured fluorescence levels using a 96-well microplate reader, a flow cytometer, as well as a confocal microscope.
                        </p>
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                    <div class="section" id="KeyFindings">
                        <h3>Key Results and Findings</h3>
                        <p>
                            Through the data obtained from our microplate reader, we found that the strengths of the three promoters investigated are in the same order as Anderson et al's initial characterization
                            across the three E. coli strains which we studied. The relative fluorescence magnitudes we measured however do not agree with
                            the ratios presented in the Anderson study.
                        </p>
                        <p>
                            To eliminate the dependence on equipment parameters associated with arbitrary fluorescence units, we calibrated our fluorescence
                            readings against different concentrations of a fluorescent chemical standard, sodium fluorescein, and expressed fluorescence
                            intensity in units of its concentration.
                        </p>
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                <div class="section" id="MethodsandMaterials">
                    <h2>Methods and Materials</h2>
                    <div class="section" id="BacterialStrainsandGrowthConditions">
                        <h3>Bacterial Strains and Growth Conditions</h3>
                        <p>
                            <i>E. coli</i> DH5α was used for all cloning purposes. <i>E. coli</i> DH5α, <i>E. coli</i> MG1655, and <i>E. coli</i> RP437
                            ∆FliC were used as expression hosts. Cultures for cloning were grown in Lysogeny Broth (LB) at 37°C, whereas for
                            GFP expression cultures were grown in <a href="http://openwetware.org/wiki/Knight:M9_supplemented_media">Knight's M9
                            Supplemented Media</a> at 37°C.
                        </p>
                    </div>
                    <div class="section" id="PlasmidConstructionsandGeneCloning">
                        <h3>Plasmid Constructions and Gene Cloning</h3>
                        <p>
                            The selected Anderson promoters were fused to the designated GFP coding sequence using the BioBrick assembly method
                            (step-by-step experimental detail as well as gel electrophoresis images can be found at the InterLab section of our lab notebook).
                        </p>
                        <p>
                            Briefly, the promoter-containing pSB1C3 plasmids <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K823005">BBa_K823005</a>,
                            <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K823008">BBa_K823008</a>,
                            <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K823013">BBa_K823013</a>,
                            and RBS-GFP-containing pSB1A2 plasmid <a href="http://parts.igem.org/Part:BBa_I13504">BBa_I13504</a> (RBS BBa_B0034 fused to GFP BBa_E0040) were obtained from the 2015
                            iGEM Distribution Kit and transformed into <i>E. coli</i> DH5α. Upon plasmid extraction (using Omega Biotek E.Z.N.A. Plasmid DNA
                            Mini Kit I, Spin Protocol according to manufacturer specifications), restriction digest was performed on the SpeI and PstI sites
                            of the promoter plasmids to linearize them, and the XbaI and PstI sites of the RBS-GFP plasmid to isolate the RBS-GFP sequence.
                            Subsequent ligations were performed to insert the isolated RBS-GFP sequence into the linearized promoter plasmids,
                            yielding the composite parts BBa_K823005+I13504, BBa_K823008+I13504, and BBa_K823013+I13504, each containing an 8bp assembly
                            scar sequence TACTAGAG between the promoter sequence and the ribosome binding site. The identities of the plasmids were
                            confirmed through sequencing (SourceBioScience, UK). The sequencing data is available for perusal in the Supplementary Information
                            section below.
                        </p>
                        <p>
                            The three composite parts were then each separately transformed into the three different <i>E. coli</i> expression host strains.
                        </p>
                    </div>
                    <div class="section" id="ExperimentalControls">
                        <h3>Experimental Controls</h3>
                        <p>
                            <a href="http://parts.igem.org/Part:BBa_I20270">BBa_I20270</a>, a constitutively-promoted GFP expression device, and <a href="http://parts.igem.org/Part:BBa_R0040">BBa_R0040</a>, a pTetR promoter with no coding sequence linked downstream were cloned into the aforementioned expression host strains to serve as positive and negative controls for the experiments respectively.
                        </p>
                    </div>
                    <div class="section" id="CalibrationChemStandard">
                        <h3>Plate Reader: Calibration against Chemical Standard</h3>
                        <p>
                            Sodium fluorescein was the chemical standard of choice for the plate reader fluorescence readings as it fluoresces in the same green colour as GFP at concentrations below 0.05M (above 0.05M, sodium fluorescein has a red tint).
                        </p>
                        <p>
                            1.66g of fluorescein (free acid) was first dissolved in 5mL of pH 8.0 Tris HCl to obtain 1M fluorescein acid solution. The solution was then neutralized with 5mL 2M NaOH (fluorescein is a dibasic acid) to obtain 10mL of 0.5M sodium fluorescein solution (hereon referred to as NaFluo).
                        </p>
                        <p>
                            Serial dilution of NaFluo was performed for the construction of calibration curve.
                        </p>
                    </div>
                    <div class="section" id="PlateReaderGrowthCond">
                        <h3>Plate Reader: Culture Growth Conditions</h3>
                        <p>
                           Stationary cultures for expression measurement were grown overnight for 18 hours
                           at 37°C in 5mL chloramphenicol-supplemented Knight's M9 Supplemented Media contained in glass test tubes oriented to be standing at an angle with
                           225 rpm orbital shaking.
                        </p>
                    </div>
                    <div class="section" id="PlateReaderSetup">
                        <h3>Plate Reader: Experimental Setup</h3>
                        <p>
                            A BMG LABTECH FLUOstar Omega was used for all plate reader measurements. For fluorescence readings, the excitation filter used was 485-12 nm and the emission filter used was 520 nm. Gain was set to 550 and number of flashes per well was set to 20. For OD<sub>600</sub> readings, the excitation filter used was 600 nm and number of flashes per well was set to 22.
                        </p>
                        <p>
                            The type of 96-well plate used was Corning® 96-well black well, clear bottom, tissue-culture treated sterile plates. 100µL of stationary culture was added into each well, and each culture was measured in triplicate. For the construction of the calibration curve, 100µL of NaFluo solution of each concentration was added to each well.
                        </p>
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                <div class="section" id="ResultsDiscussion">
                    <h2>Results and Discussion</h2>
                    <div class="section" id="Calibration">
                        <h3>Calibration against Sodium Fluorescein</h3>
                        <p>
                            <div class="image image-right">
                                <img src="https://static.igem.org/mediawiki/2015/2/21/Oxford15_interlabcalicur.png" width="433" height="400" />
                                <p>PCR Results</p>
                            </div>
                        </p>
                    </div>
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        <div class="col-md-3 contents-sidebar">
            <ul id="sidebar" class="nav nav-stacked" data-spy="affix">
                <li>
                    <a href="#Introduction">Introduction</a>
                	<ul class="nav nav-stacked">
                        <li><a href="#Overview">Overview</a></li>
                        <li><a href="#KeyFindings">Key Results and Findings</a></li>
                    </ul>
                    <a href="#MethodsandMaterials">Methods and Materials</a>
                    <ul class="nav nav-stacked">
                        <li><a href="#BacterialStrainsandGrowthConditions">Bacterial Strains and Growth Conditions</a></li>
                        <li><a href="#PlasmidConstructionsandGeneCloning">Plasmid Construction and Gene Cloning</a></li>
                        <li><a href="#ExperimentalControls">Experimental Controls</a></li>
                        <li><a href="#CalibrationChemStandard">Plate Reader: Calibration against Chemical Standard</a></li>
                        <li><a href="#PlateReaderGrowthCond">Plate Reader: Culture Growth Conditions</a></li>
                        <li><a href="#PlateReaderSetup">Plate Reader: Experimental Setup</a></li>
                    </ul>
                    <a href="#ResultsDiscussion">Results and Discussion</a>
                    <ul class="nav nav-stacked">
                        <li><a href="#Calibration">Calibration against Sodium Fluorescein</a></li>
                    </ul>
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