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| − | </script>
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| − | </body>
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| − | </html>
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| − | <div class="grid-sixcolumns">
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| − | <div class="button_grid_sixcolumns">
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| − | <a style="display: block; width: 100%;" href="#Overview"><button class="button">Overview</button></a>
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| − | </div>
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| − | <div class="button_grid_sixcolumns">
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| − | <a style="display: block; width: 100%;" href="#Overexpression"><button class="button">Overexpression</button></a>
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| − | </div>
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| − | <div class="button_grid_sixcolumns">
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| − | <a style="display: block; width: 100%;" href="#Biosensor"> <button class="button">Biosensor</button></a>
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| − | </div>
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| − | <div class="filler"></div>
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| − | <div class="button_grid_sixcolumns">
| |
| − | <a style="display: block; width: 100%;" href="#Integration"><button class="button">Integration</button></a>
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| − | </div>
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| − | <div class="button_grid_sixcolumns">
| |
| − | <a style="display: block; width: 100%;" href="#Conclusion"><button class="button">Conclusion</button></a>
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| − | </div>
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| − | <div class="filler"></div>
| |
| − | </div>
| |
| − |
| |
| − | <a name="Overeview">
| |
| − | <p class="subapart1">Overview</p>
| |
| − | </a>
| |
| − | <ul style="background-color: #c6d1e5; border-radius: 15px; padding: 1.8rem; max-width: 100%">
| |
| − | <li style="padding: 0.3rem">When fadL and fadD are overexpressed LCFA uptake increases resulting in a higher
| |
| − | growth if LCFA are the only carbon source available. </li>
| |
| − | <li style="padding: 0.3rem">Lorem ipsum dolor sit amet consectetur adipisicing elit. Natus, recusandae.</li>
| |
| − | <li style="padding: 0.3rem">Lorem ipsum dolor sit amet consectetur adipisicing elit. Aperiam quas, cumque
| |
| − | obcaecati iste vero facilis eaque mollitia animi similique debitis.</li>
| |
| − | </ul>
| |
| − |
| |
| − | <a name="Overexpression">
| |
| − | <p class="subapart1">Overexpression results</p>
| |
| − | </a>
| |
| − | <p>In order to study the functionality of our system we characterized the constructs both in palmitic acid (PA)
| |
| − | and oleic acid (OA). Bacteria was grown for 3 days in M9 minimal medium with the respective fatty acid, so as
| |
| − | to be sure that the bacteria was only using LCFA as a carbon source. Additionally, the supernatant was also
| |
| − | collected and LCFA concentration was measured using cupric-acetate colorimetric (see <a href="https://2018.igem.org/Team:UPF_CRG_Barcelona/Methods">Methods
| |
| − | </a>) technique in OD715nm. This same growth assay was also performed in LB to study growth in enriched
| |
| − | media.</p> <p>Different levels of fad genes expression were achieved through different strains. The genetic
| |
| − | constructs consisting of either FadD or FadL downstream the TetR repressible promoter were transformed in
| |
| − | top10 E. coli strain (DH5-alpha) and Zn1. Top 10 constitutively synthesize tetR, which results in a
| |
| − | continuous expression of our construct. Zn1 does not express tetR, thus meaning that our genetic constructs
| |
| − | will not be induced. This induction can be modulated by adding anhydrotetracycline (ATc) in the medium. We
| |
| − | created a cell library expressing the fadD and fadL proteins: </p>
| |
| − | <p class="subapart1">FadD overexpression increases growth when either PA or OA are the only carbon source. </p>
| |
| − |
| |
| − | <p>We analyzed the behavior of the constitutive and inducible FadD cell lines with M9 minimal media
| |
| − | supplemented with either PA or OA. Constitutive reporter cell line was used as a control. OD measurements
| |
| − | were performed at OD 600 nm as an indicative cell growth for 72 hours.</p>
| |
| − |
| |
| − | <p>Our results show that, when fadD is overexpressed, bacterial growth increases both in 0,4 mM PA (Fig xA) and
| |
| − | 2 mM PA (figure Xb) in relation to control. Moreover, the non-induced FadD cell show a lower increase of
| |
| − | bacterial growth, compared with the control. On the other hand, when tested with OA medium (fig Xc and d),
| |
| − | the difference in growth is higher than in PA medium. </p>
| |
| − |
| |
| − | <p class="fig-caption"><b>Figure X | Growth assay of induced and not induced fadD in minimal medium enriched
| |
| − | with different concentrations of Oleic Acid (OA) and Palmitic Acid (PA).</b> Figure A show OD600nm over
| |
| − | time of induced (top10 strain) and not induced (Zn1 strain) fadD in 0,4mM PA concentration. Figure B show
| |
| − | OD600nm over time of induced (top10 strain) and not induced (Zn1 strain) fadD in 2mM PA concentration. Figure
| |
| − | C show OD600nm over time of induced (top10 strain) and not induced (Zn1 strain) fadD in 0,4mM OA
| |
| − | concentration. Figure D show OD600nm over time of induced (top10 strain) and not induced (Zn1 strain) fadD in
| |
| − | 2mM OA concentration. Error bars represent the Standard Deviation of the Mean (SEM).</p>
| |
| − |
| |
| − | <p>Moreover, growth in an enriched media (LB) was studied. Expression of induced FadD cell line was tuned with
| |
| − | ATc. Tuneable FadD overexpression levels entailed a big metabolic burden (figure X). Our results show that
| |
| − | when ATc induction increases, growth rate diminishes. In this way, our results suggested that even when PA is
| |
| − | available in the media, our system will prefer other carbon sources. However, when LCFA are the only ones
| |
| − | being present, the overexpression of fad genes gives an advantage reflected in enhanced growth (figure X).
| |
| − | </p>
| |
| − |
| |
| − | <p class="fig-caption"><b>Figure X | Growth assay of induced and not induced fadD in enriched medium. </b>
| |
| − | Bacteria was grown for 16 hours in enriched medium with a concentration of 0,4mM Palmitic Acid. All bacteria
| |
| − | was top10 (DH5-alpha strain). To induce fadD expression a concentration of 60ng/ml of ATc was added. Error
| |
| − | bars represent the Standard Deviation of the Mean (SEM).</p>
| |
| − |
| |
| − | <p class="subapart1">FadL overexpression reduces metabolic burden when both PA and OA concentration increases.</p>
| |
| − | <p>We analyzed the behavior of the constitutive and inducible FadL cell lines with M9 minimal media
| |
| − | supplemented with either PA or OA. Constitutive reporter cell line was used as a control. OD measurements
| |
| − | were performed at OD 600nm as an indicative cell growth for 72 hours.</p>
| |
| − |
| |
| − | <p>Here we demonstrate that overexpression of fadL entails a metabolic burden. This is shown in the decrease of
| |
| − | the OD600 when induced bacteria is grown in both 0,4mM PA or OA (figure X) and 2mM PA or OA (figure X). When
| |
| − | comparing this metabolic burden between the two concentrations it is clear that in the 2mM concentration
| |
| − | induced bacteria grow more than in 0,4mM. Thus, this results suggest that FadL entails a metabolic burden
| |
| − | even in minimum media but is reduced when LCFA concentration increases. </p>
| |
| − |
| |
| − | <p class="fig-caption"><b>Figure X. Growth assay of induced and not induced fadL in minimal medium enriched
| |
| − | with different concentrations of Oleic Acid (OA) and Palmitic Acid (PA). </b>Figure A show OD600nm over
| |
| − | time of induced (top10 strain) and not induced (Zn1 strain) fadL in 0,4mM PA concentration. Figure B show
| |
| − | OD600nm over time of induced (top10 strain) and not induced (Zn1 strain) fadL in 2mM PA concentration. Figure
| |
| − | C show OD600nm over time of induced (top10 strain) and not induced (Zn1 strain) fadL in 0,4mM OA
| |
| − | concentration. Figure D show OD600nm over time of induced (top10 strain) and not induced (Zn1 strain) fadL in
| |
| − | 2mM OA concentration. Error bars represent the Standard Deviation of the Mean (SEM).</p>
| |
| − |
| |
| − | <p class="subapart1">FadD overexpression increases OA uptake</p>
| |
| − | <p>We analyzed the behavior of the constitutive and inducible FadD cell lines with M9 minimal media
| |
| − | supplemented with either PA or OA. Constitutive reporter cell line was used as a control. Supernatant was
| |
| − | collected from the medium after 72 hours growing. Cupric-acetate colorimetric technique was performed
| |
| − | quantify LCFA concentration (OD715nm).</p>
| |
| − |
| |
| − | <p>Our results showed that, when fadD was overexpressed, OA uptake nearly doubled the uptake of non induced
| |
| − | bacteria and control (p < 0.001) (Fig. X). However, this increase could not be observed in PA. A possible
| |
| − | explanation would be the infeasibility to obtain a proper standard curve for PA using cupric-acetate
| |
| − | technique when compared to oleic acid (see methods). </p>
| |
| − | <div id="results_overexpression1" style="max-width: 70vw;">
| |
| − | </div>
| |
| − | <script>
| |
| − | var control = {
| |
| − | x: ['Palmitic Acid', 'Oleic Acid'],
| |
| − | y: [0.059049989, 0.015975604],
| |
| − | name: 'Control',
| |
| − | type: 'bar',
| |
| − | error_y: {
| |
| − | type: 'data',
| |
| − | array: [0.00098995, 0.002899139],
| |
| − | visible: true,
| |
| − | thickness: 1,
| |
| − | color: '#36393D',
| |
| − | },
| |
| − | width: .2,
| |
| − | marker: {
| |
| − | color: '#0079bf',
| |
| − | }
| |
| − | };
| |
| − | var induced = {
| |
| − | x: ['Palmitic Acid', 'Oleic Acid'],
| |
| − | y: [0.051237715, 0.027976643],
| |
| − | name: 'Induced',
| |
| − | type: 'bar',
| |
| − | marker: {
| |
| − | color: "#557d95",
| |
| − | },
| |
| − | width: .2,
| |
| − | error_y: {
| |
| − | type: 'data',
| |
| − | array: [0.002687004, 0.000636396],
| |
| − | visible: true,
| |
| − | thickness: 1,
| |
| − | color: '#36293D',
| |
| − | },
| |
| − | };
| |
| − | var notinduced = {
| |
| − | x: ['Palmitic Acid', 'Oleic Acid'],
| |
| − | y: [0.048294151, 0.021051573],
| |
| − | name: 'Not induced',
| |
| − | type: 'bar',
| |
| − | marker: {
| |
| − | color: "#225a7f",
| |
| − | },
| |
| − | width: .2,
| |
| − | error_y: {
| |
| − | type: 'data',
| |
| − | array: [0.000353554107089698, 0.0000707066067330886],
| |
| − | visible: true,
| |
| − | thickness: 1,
| |
| − | color: '#36393D',
| |
| − | },
| |
| − | };
| |
| − | var data = [induced, notinduced, control];
| |
| − | var layout = {
| |
| − | barmode: 'group',
| |
| − | title: "FadD",
| |
| − | titlefont: {
| |
| − | family: "PT Sans",
| |
| − | size: 15,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | xaxis: {
| |
| − | autorange: true,
| |
| − | showticklabels: true,
| |
| − | tickfont: {
| |
| − | family: "PT Sans",
| |
| − | size: 13,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | },
| |
| − | yaxis: {
| |
| − | zeroline: false,
| |
| − | autorange: true,
| |
| − | title: "something",
| |
| − | titlefont: {
| |
| − | family: "PT Sans",
| |
| − | size: 13,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | showticklabels: true,
| |
| − | tickfont: {
| |
| − | family: "PT Sans",
| |
| − | size: 11,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | },
| |
| − | };
| |
| − | Plotly.newPlot('results_overexpression1', data, layout, {
| |
| − | displayModeBar: false
| |
| − | });
| |
| − | </script>
| |
| − | <p class="fig-caption"><b>Figure X | LCFA uptake in FadD overexpression. </b>Plot showing LCFA uptake when
| |
| − | comparing induced (Top10 strain) and not induced (Zn1 strain) fadD gene. Constitutive reporter cell line
| |
| − | (top10) was used as a control. Uptake was measured considering the OD715nm difference between the medium at
| |
| − | certain concentration of LCFA and the medium with grown bacteria. A concentration of 0,4mM of both PA and OA
| |
| − | was used. Cupric-acetate technique was used to quantify LCFA in the medium. LCFA uptake was normalized by the
| |
| − | growth of the bacteria, measured at OD600. Error bars represent the Standard Deviation of the Mean (SEM).
| |
| − | Statistical significance of the mean was calculated using a paired t-test. P value < 0,05, which indicates a
| |
| − | statistically significant difference among relevant groups, is designated with an asterisk.</p> <p class="subapart1">Overexpression
| |
| − | of fadL increases OA uptake.</p>
| |
| − | <p>Here to study the LCFA uptake the same experimental procedure was followed as described in FadD. Our results
| |
| − | show that, when fadL was induced OA uptake was more than 2-fold higher in relation to non-induced and control
| |
| − | bacteria (Fig. X). This increased uptake was only statistically significant in OA when compared to control (p < 0.0001)
| |
| − | and not induced bacteria (p < 0.0001). PA media didn’t show significant results (figure X).</p> <div id="results_overexpression2"
| |
| − | style="max-width: 70vw;">
| |
| − | </div>
| |
| − | <script>
| |
| − | var control = {
| |
| − | x: ['Palmitic Acid', 'Oleic Acid'],
| |
| − | y: [0.059049989, 0.015975604],
| |
| − | name: 'Control',
| |
| − | type: 'bar',
| |
| − | width: .2,
| |
| − | error_y: {
| |
| − | type: 'data',
| |
| − | array: [0.00098995, 0.002899139],
| |
| − | visible: true,
| |
| − | thickness: 1,
| |
| − | color: '#36393D',
| |
| − | },
| |
| − | marker: {
| |
| − | color: '#0079bf',
| |
| − | }
| |
| − | };
| |
| − | var induced = {
| |
| − | x: ['Palmitic Acid', 'Oleic Acid'],
| |
| − | y: [0.053295016, 0.035822019],
| |
| − | name: 'Induced',
| |
| − | type: 'bar',
| |
| − | width: .2,
| |
| − | marker: {
| |
| − | color: "#557d95",
| |
| − | },
| |
| − | error_y: {
| |
| − | type: 'data',
| |
| − | array: [0.000424261, 0.001697059],
| |
| − | visible: true,
| |
| − | thickness: 1,
| |
| − | color: '#36293D',
| |
| − | },
| |
| − | };
| |
| − | var notinduced = {
| |
| − | x: ['Palmitic Acid', 'Oleic Acid'],
| |
| − | y: [0.053694714, 0.014226389],
| |
| − | name: 'Not induced',
| |
| − | type: 'bar',
| |
| − | width: .2,
| |
| − | marker: {
| |
| − | color: "#225a7f",
| |
| − | },
| |
| − | error_y: {
| |
| − | type: 'data',
| |
| − | array: [0.002687004, 0.000989945],
| |
| − | visible: true,
| |
| − | thickness: 1,
| |
| − | color: '#36393D',
| |
| − | },
| |
| − | };
| |
| − | var data = [induced, notinduced, control];
| |
| − | var layout = {
| |
| − | barmode: 'group',
| |
| − | title: "FadL",
| |
| − | titlefont: {
| |
| − | family: "PT Sans",
| |
| − | size: 15,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | xaxis: {
| |
| − | autorange: true,
| |
| − | showticklabels: true,
| |
| − | tickfont: {
| |
| − | family: "PT Sans",
| |
| − | size: 13,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | },
| |
| − | yaxis: {
| |
| − | zeroline: false,
| |
| − | autorange: true,
| |
| − | title: "SOMETHING",
| |
| − | titlefont: {
| |
| − | family: "PT Sans",
| |
| − | size: 13,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | showticklabels: true,
| |
| − | tickfont: {
| |
| − | family: "PT Sans",
| |
| − | size: 11,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | },
| |
| − | };
| |
| − | Plotly.newPlot('results_overexpression2', data, layout, {
| |
| − | displayModeBar: false
| |
| − | });
| |
| − | </script>
| |
| − |
| |
| − | <p class="fig-caption"><b>Figure X. LCFA uptake in FadL overexpression.</b> Plot showing LCFA uptake when
| |
| − | comparing induced (Top10 strain) and not induced (Zn1 strain) fadL gene. Constitutive reporter cell line
| |
| − | (top10) was used as a control. Uptake was measured considering the OD715nm difference between the medium at
| |
| − | certain concentration of LCFA and the medium with grown bacteria. A concentration of 0,4mM of both PA and
| |
| − | OA was used. Cupric-acetate technique was used to quantify LCFA in the medium. LCFA uptake was normalized
| |
| − | by the growth of the bacteria, measured at OD600. Error bars represent the Standard Deviation of the Mean
| |
| − | (SEM). Statistical significance of the mean was calculated using a paired t-test. P value < 0.05, which
| |
| − | indicates a statistically significant difference among relevant groups, is designated with an asterisk.</p>
| |
| − | <p class="subapart1">Discussion: </p>
| |
| − | <p>Our results demonstrate that overexpression of fadL and fadD in enriched media entails a metabolic burden
| |
| − | for the cell. However, when these genes are overexpressed in a minimum media, with LCFA as the unique
| |
| − | carbon sources, the metabolic burden is reduced. Furthermore, bacterial growth is even higher between
| |
| − | induced and not induced cells. We have demonstrated that induction of fadL and fadD results in a LCFA
| |
| − | uptake increase, being statistically significant in OA. </p>
| |
| − |
| |
| − | <p>Considering our results, it can be deduced that when fadL and fadD are overexpressed LCFA uptake increases
| |
| − | resulting in a higher growth. In this way, we can conclude that LCFA influx is increased in our system,
| |
| − | resulting in higher Acyl-CoA concentration inside the cell. Consequently, we have hypothesized that this
| |
| − | results in an enhanced expression of the rest of fad genes and therefore in an increase of the LCFA
| |
| − | degradation rate. This increase in the LCFA degradation, leads to more metabolic fuel to be used for growth
| |
| − | (only when LCFA is the only carbon source available). Therefore, overexpression of fadL and fadD gives the
| |
| − | cell a positive advantage that results in enhanced growing when LCFA is the only carbon source. </p>
| |
| − |
| |
| − | <a name="Biosensor">
| |
| − | <p class="subapart1">Biosensor Results</p>
| |
| − | </a>
| |
| − | <p class="subapart2">Characterization of the Fatty acid acyl-CoA biosensor (pFadBA)</p>
| |
| − | <p>To evaluate the function of the pfadBA promoter, we designed a reporter system with RFP. TOP-10 <i>E. coli</i>
| |
| − | strain expressing the construct were induced with different concentrations of PA in LB medium. Fluorescence
| |
| − | was
| |
| − | analyzed once it had reached the steady state.</p>
| |
| − |
| |
| − | <div id="results_biosensor1" style="max-width: 70vw;"></div>
| |
| − | <script>
| |
| − | var trace1 = {
| |
| − | x: [
| |
| − | 0, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1
| |
| − | ],
| |
| − | y: [
| |
| − | 166.1957236, 170.9917908, 180.3131834, 183.0117334, 188.0914255, 208.3651179, 221.8960085,
| |
| − | 256.4273664,
| |
| − | ],
| |
| − | error_y: {
| |
| − | color: '#0079bf',
| |
| − | thickness: 1,
| |
| − | type: 'data',
| |
| − | array: [
| |
| − | 18.02107414, 18.15429603, 11.91156693, 6.577895472, 7.200809337, 11.55551043, 14.45362679,
| |
| − | 18.01708433,
| |
| − | ],
| |
| − | visible: true
| |
| − | },
| |
| − | type: 'scatter',
| |
| − | line: {
| |
| − | color: "#0079bf",
| |
| − | width: 1,
| |
| − | dash: "dot",
| |
| − | },
| |
| − | };
| |
| − | var data = [trace1];
| |
| − | var layout = {
| |
| − | title: "Mean Fluorescence Intensity",
| |
| − | titlefont: {
| |
| − | family: "PT Sans Bold",
| |
| − | size: 16,
| |
| − | },
| |
| − | xaxis: {
| |
| − | title: "Palmitic Acid concentration (mM)",
| |
| − | titlefont: {
| |
| − | family: "PT Sans",
| |
| − | size: 15,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | showticklabels: true,
| |
| − | tickfont: {
| |
| − | family: "PT Sans",
| |
| − | size: 12,
| |
| − | color: "#36393d",
| |
| − |
| |
| − | },
| |
| − | },
| |
| − | yaxis: {
| |
| − | title: "Fluorescence (a.u.)",
| |
| − | titlefont: {
| |
| − | family: "PT Sans",
| |
| − | size: 15,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | showticklabels: true,
| |
| − | tickfont: {
| |
| − | family: "PT Sans",
| |
| − | size: 12,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | },
| |
| − | };
| |
| − | Plotly.newPlot('results_biosensor1', data, layout, {
| |
| − | displayModeBar: false
| |
| − | });
| |
| − | </script>
| |
| − | <p>Our data indicates that the activation threshold of the pfadBA promoter is at 0.2 mM of PA. The baseline
| |
| − | fluorescence is really high and we only observed a fold change of 1.5 between the fluorescence of the non
| |
| − | induced bacteria and of the maximum induced one (PA 1mM). </p>
| |
| − | <p>We could not see the saturation in the transfer function due to the fact that medium with PA
| |
| − | concentrations
| |
| − | higher than 1 mM created a lot of noise which made impossible to analyse fluorescence at this high
| |
| − | concentrations. </p>
| |
| − |
| |
| − | <p class="subapart2">Characterization of the Inducible LuxR-pLux engineered device</p>
| |
| − |
| |
| − | <p>In order to modulate the expression of the genes expressed under the pfadBA promoter, we designed a pfadBA
| |
| − | reporter system inducible with both LCFA and lactone. TOP 10 bacteria expressing the construct were induced
| |
| − | with different concentrations of lactone and 3 different concentrations of PA (0, 0.4 and 1 mM) in LB
| |
| − | medium.
| |
| − | Fluorescence was analyzed once it had reached the steady state.</p>
| |
| − |
| |
| − | <div id="results_biosensor2" style="max-width: 70vw;"></div>
| |
| − | <script>
| |
| − | var trace0 = {
| |
| − | x: [0, 0.0000000001, 0.000000001, 0.00000005, 0.0000001, 0.0000005],
| |
| − | y: [0, 0, 0, 44.56481895, 170.446603, 215.0904724, 204.0716625],
| |
| − | error_y: {
| |
| − | color: '#0079bf',
| |
| − | thickness: 1,
| |
| − | type: 'data',
| |
| − | array: [0, 0, 0, 15.29557911, 19.73763307, 17.61577669, 28.03661507],
| |
| − | visible: true
| |
| − | },
| |
| − | type: 'scatter',
| |
| − | line: {
| |
| − | color: "#557d95",
| |
| − | width: 1,
| |
| − | dash: "dot",
| |
| − | hape: "spline",
| |
| − | },
| |
| − | name: "LB",
| |
| − | };
| |
| − | var trace1 = {
| |
| − | x: [0, 0.0000000001, 0.000000001, 0.00000005, 0.0000001, 0.0000005],
| |
| − | y: [0, 0, 0, 82.84644574, 191.2132307, 259.1304718, 249.2150203],
| |
| − | error_y: {
| |
| − | color: '#0079bf',
| |
| − | thickness: 1,
| |
| − | type: 'data',
| |
| − | array: [0, 0, 0.838338218, 9.309595929, 12.83295675, 8.806343893, 25.20344811],
| |
| − | visible: true
| |
| − | },
| |
| − | type: 'scatter',
| |
| − | line: {
| |
| − | color: "#0079bf",
| |
| − | width: 1,
| |
| − | dash: "dot",
| |
| − | shape: "spline",
| |
| − | },
| |
| − | name: "LB + 0.4 mM Palmitic Acid",
| |
| − | };
| |
| − | var trace2 = {
| |
| − | x: [0, 0.0000000001, 0.000000001, 0.00000005, 0.0000001, 0.0000005],
| |
| − | y: [0, 0, 0, 59.81279408, 242.2969439, 351.8179998, 352.8631549],
| |
| − | error_y: {
| |
| − | color: '#225A7F',
| |
| − | thickness: 1,
| |
| − | type: 'data',
| |
| − | array: [0.659347939, 0.48185037, 1.213116806, 12.08067271, 48.00392739, 26.8915842, 27.55701065],
| |
| − | visible: true
| |
| − | },
| |
| − | type: 'scatter',
| |
| − | line: {
| |
| − | color: "#225A7F",
| |
| − | width: 1,
| |
| − | dash: "dot",
| |
| − | hape: "spline",
| |
| − | },
| |
| − | name: "LB + 1 mM Palmitic Acid",
| |
| − | };
| |
| − | var data = [trace0, trace1, trace2];
| |
| − | var layout = {
| |
| − | title: "Mean Fluorescence Intensity",
| |
| − | titlefont: {
| |
| − | family: "PT Sans Bold",
| |
| − | size: 16,
| |
| − | },
| |
| − | xaxis: {
| |
| − | type: "log",
| |
| − | exponentformat: 'e',
| |
| − | showexponent: 'all',
| |
| − | title: "Lactone Concentration (M)",
| |
| − | titlefont: {
| |
| − | family: "PT Sans",
| |
| − | size: 15,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | showticklabels: true,
| |
| − | tickfont: {
| |
| − | family: "PT Sans",
| |
| − | size: 12,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | },
| |
| − | yaxis: {
| |
| − | title: "Fluorescence (a.u.)",
| |
| − | titlefont: {
| |
| − | family: "PT Sans",
| |
| − | size: 15,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | showticklabels: true,
| |
| − | tickfont: {
| |
| − | family: "PT Sans",
| |
| − | size: 12,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | },
| |
| − | };
| |
| − | Plotly.newPlot('results_biosensor2', data, layout, {
| |
| − | displayModeBar: false
| |
| − | });
| |
| − | </script>
| |
| − |
| |
| − | <p>Our data indicates that the activation threshold of this lactone inducible pfafba construct is
| |
| − | approximately
| |
| − | at 5x10<sup>-9</sup> M of lactone. It saturates at 1x10<sup>-7</sup> M of lactone. Significant fluorescence
| |
| − | difference between different PA concentrations is only observed at saturated lactone ranges. </p>
| |
| − |
| |
| − | <p class="subapart2">Characterization of the cI mediated activity</p>
| |
| − |
| |
| − | <div id="results_biosensor3" style="max-width: 70vw;"></div>
| |
| − | <script>
| |
| − | var trace1 = {
| |
| − | x: [
| |
| − | 0, 0.5, 1, 1.5
| |
| − | ],
| |
| − | y: [
| |
| − | 14.87981941, 27.13469968, 46.66896681, 63.57392764
| |
| − | ],
| |
| − | error_y: {
| |
| − | color: '#0079bf',
| |
| − | thickness: 1,
| |
| − | type: 'data',
| |
| − | array: [5.254043865, 3.122421295, 4.348284106, 5.052001885, ],
| |
| − | visible: true
| |
| − | },
| |
| − | type: 'scatter',
| |
| − | line: {
| |
| − | color: "#0079bf",
| |
| − | width: 1,
| |
| − | dash: "dot",
| |
| − | },
| |
| − | };
| |
| − | var data = [trace1];
| |
| − | var layout = {
| |
| − | title: "Mean Fluorescence Intensity",
| |
| − | titlefont: {
| |
| − | family: "PT Sans Bold",
| |
| − | size: 16,
| |
| − | },
| |
| − | xaxis: {
| |
| − | title: "Palmitic Acid concentration (mM)",
| |
| − | titlefont: {
| |
| − | family: "PT Sans",
| |
| − | size: 15,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | showline: true,
| |
| − | showticklabels: true,
| |
| − | tickfont: {
| |
| − | family: "PT Sans",
| |
| − | size: 12,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | },
| |
| − | yaxis: {
| |
| − | title: "Fluorescence (a.u.)",
| |
| − | titlefont: {
| |
| − | family: "PT Sans",
| |
| − | size: 15,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | showticklabels: true,
| |
| − | tickfont: {
| |
| − | family: "PT Sans",
| |
| − | size: 12,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | },
| |
| − | };
| |
| − | Plotly.newPlot('results_biosensor3', data, layout, {
| |
| − | displayModeBar: false
| |
| − | });
| |
| − | </script>
| |
| − | <p class="subapart2">Comparative study between the Lux, the pFadBA and the cI biosensors</p>
| |
| − |
| |
| − | <div id="results_biosensor4" style="max-width: 70vw;"></div>
| |
| − | <script>
| |
| − | var pFadBA = {
| |
| − | x: ['LB', 'LB 0.4 mM PA', 'LB 1 mM PA'],
| |
| − | y: [166.1957236, 188.0914255, 256.4273664],
| |
| − | name: 'pFadBa',
| |
| − | type: 'bar',
| |
| − | error_y: {
| |
| − | type: 'data',
| |
| − | array: [18.02107414, 7.200809337, 18.01708433],
| |
| − | visible: true,
| |
| − | thickness: 1,
| |
| − | color: '#36393D',
| |
| − | },
| |
| − | width: .2,
| |
| − | marker: {
| |
| − | color: '#0079bf',
| |
| − | }
| |
| − | };
| |
| − | var lux = {
| |
| − | x: ['LB', 'LB 0.4 mM PA', 'LB 1 mM PA'],
| |
| − | y: [212.9574509, 262.1183264, 351.8179998],
| |
| − | name: 'Inducible LuxR-pLux ',
| |
| − | type: 'bar',
| |
| − | error_y: {
| |
| − | type: 'data',
| |
| − | array: [16.52376544, 23.85503417, 26.8915842],
| |
| − | visible: true,
| |
| − | thickness: 1,
| |
| − | color: '#36393D',
| |
| − | },
| |
| − | width: .2,
| |
| − | marker: {
| |
| − | color: '#225a7f',
| |
| − | }
| |
| − | };
| |
| − | var ci = {
| |
| − | x: ['LB', 'LB 0.4 mM PA', 'LB 1 mM PA'],
| |
| − | y: [14.87981941, 27.13469968, 46.66896681],
| |
| − | name: 'HighPass Filter',
| |
| − | type: 'bar',
| |
| − | error_y: {
| |
| − | type: 'data',
| |
| − | array: [5.254043865, 3.122421295, 4.348284106],
| |
| − | visible: true,
| |
| − | thickness: 1,
| |
| − | color: '#36393D',
| |
| − | },
| |
| − | width: .2,
| |
| − | marker: {
| |
| − | color: '#577d95',
| |
| − | }
| |
| − | };
| |
| − | var data = [lux, pFadBA, ci];
| |
| − | var layout = {
| |
| − | barmode: 'group',
| |
| − | title: "Comparison of the fluorescence",
| |
| − | titlefont: {
| |
| − | family: "PT Sans Bold",
| |
| − | size: 16,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | xaxis: {
| |
| − | title: "",
| |
| − | titlefont: {
| |
| − | size: 15,
| |
| − | },
| |
| − | autorange: true,
| |
| − | showticklabels: true,
| |
| − | tickfont: {
| |
| − | family: "PT Sans",
| |
| − | size: 12,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | },
| |
| − | yaxis: {
| |
| − | zeroline: false,
| |
| − | autorange: true,
| |
| − | title: "Fluorescence (a.u.)",
| |
| − | titlefont: {
| |
| − | family: "PT Sans",
| |
| − | size: 15,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | showticklabels: true,
| |
| − | tickfont: {
| |
| − | family: "PT Sans",
| |
| − | size: 12,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | },
| |
| − | };
| |
| − | Plotly.newPlot('results_biosensor4', data, layout, {
| |
| − | displayModeBar: false
| |
| − | });
| |
| − | </script>
| |
| − | <p class="subapart2">Characterization of the Improved fatty acid acyl-CoA biosensor (pAR)</p>
| |
| − |
| |
| − | <div id="results_biosensor5" style="max-width: 70vw;"></div>
| |
| − | <script>
| |
| − | var pFadBA = {
| |
| − | x: ['LB', 'LB 0.4 mM PA', 'LB 1 mM PA'],
| |
| − | y: [166.1957236, 188.0914255, 256.4273664],
| |
| − | name: 'pFadBa',
| |
| − | type: 'bar',
| |
| − | width: .3,
| |
| − | error_y: {
| |
| − | type: 'data',
| |
| − | array: [18.02107414, 7.200809337, 18.01708433],
| |
| − | visible: true,
| |
| − | thickness: 1,
| |
| − | color: '#36393D',
| |
| − | },
| |
| − | marker: {
| |
| − | color: '#0079bf',
| |
| − | }
| |
| − | };
| |
| − | var par = {
| |
| − | x: ['LB', 'LB 0.4 mM PA', 'LB 1 mM PA'],
| |
| − | y: [54.71187136, 155.836331, 175.0262161],
| |
| − | name: 'pAR',
| |
| − | type: 'bar',
| |
| − | width: .3,
| |
| − | error_y: {
| |
| − | type: 'data',
| |
| − | array: [4.129282683, 3.299322183, 8.466116857],
| |
| − | visible: true,
| |
| − | thickness: 1,
| |
| − | color: '#36393D',
| |
| − | },
| |
| − | marker: {
| |
| − | color: '#225a7f',
| |
| − | }
| |
| − | };
| |
| − | var data = [pFadBA, par];
| |
| − | var layout = {
| |
| − | barmode: 'group',
| |
| − | title: "Comparison of fluorescence between pFadBA and pAR",
| |
| − | titlefont: {
| |
| − | family: "PT Sans Bold",
| |
| − | size: 16,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | xaxis: {
| |
| − | title: "",
| |
| − | titlefont: {
| |
| − | size: 15,
| |
| − | },
| |
| − | autorange: true,
| |
| − | showticklabels: true,
| |
| − | tickfont: {
| |
| − | family: "PT Sans",
| |
| − | size: 12,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | },
| |
| − | yaxis: {
| |
| − | zeroline: false,
| |
| − | autorange: true,
| |
| − | title: "Fluorescence (a.u.)",
| |
| − | titlefont: {
| |
| − | family: "PT Sans",
| |
| − | size: 15,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | showticklabels: true,
| |
| − | tickfont: {
| |
| − | family: "PT Sans",
| |
| − | size: 12,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | },
| |
| − | };
| |
| − | Plotly.newPlot('results_biosensor5', data, layout, {
| |
| − | displayModeBar: false
| |
| − | });
| |
| − | </script>
| |
| − | <div id="results_biosensor6" style="max-width: 70vw;"></div>
| |
| − | <script>
| |
| − | var pFadBA = {
| |
| − | x: ['LB', 'LB 0.4 mM PA', 'LB 1 mM PA'],
| |
| − | y: [1, 1.131746482, 1.542923974],
| |
| − | name: 'pFadBa',
| |
| − | type: 'bar',
| |
| − | width: .3,
| |
| − | marker: {
| |
| − | color: '#0079bf',
| |
| − | }
| |
| − | };
| |
| − |
| |
| − | var pAR = {
| |
| − | x: ['LB', 'LB 0.4 mM PA', 'LB 1 mM PA'],
| |
| − | y: [1, 2.848309281, 3.19905373],
| |
| − | name: 'pAR',
| |
| − | type: 'bar',
| |
| − | width: .3,
| |
| − | marker: {
| |
| − | color: '#225a7f',
| |
| − | }
| |
| − | };
| |
| − |
| |
| − | var data = [pFadBA, pAR];
| |
| − | var layout = {
| |
| − | barmode: 'group',
| |
| − | title: "Comparison of Fold Change between pFadBA and pAR",
| |
| − | titlefont: {
| |
| − | family: "PT Sans Bold",
| |
| − | size: 16,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | xaxis: {
| |
| − | title: "",
| |
| − | titlefont: {
| |
| − | size: 15,
| |
| − | },
| |
| − | autorange: true,
| |
| − | showticklabels: true,
| |
| − | tickfont: {
| |
| − | family: "PT Sans",
| |
| − | size: 12,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | },
| |
| − | yaxis: {
| |
| − | zeroline: false,
| |
| − | autorange: true,
| |
| − | title: "Fold Change (FC/LB)",
| |
| − | titlefont: {
| |
| − | family: "PT Sans",
| |
| − | size: 15,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | showticklabels: true,
| |
| − | tickfont: {
| |
| − | family: "PT Sans",
| |
| − | size: 12,
| |
| − | color: "#36393d",
| |
| − | },
| |
| − | },
| |
| − | };
| |
| − | Plotly.newPlot('results_biosensor6', data, layout, {
| |
| − | displayModeBar: false
| |
| − | });
| |
| − | </script>
| |
| − | <a name="Integration">
| |
| − | <p class="subapart1">Integration Results</p>
| |
| − | </a>
| |
| − | <a name="Conclusions">
| |
| − | <p class="subapart1">Conclusions</p>
| |
| − | </a>
| |
| − |
| |
| − |
| |
| − |
| |
| − | </div>
| |
| − |
| |
| − | </section>
| |
| − |
| |
| − | </main>
| |
| − |
| |
| − | <div class="spacer"></div>
| |
| − |
| |
| − | </html>
| |
| − |
| |
| − | {{UPF_CRG_Barcelona/footer}}
| |
| − |
| |
| − | <html>
| |
| − | <!-- JS code -->
| |
| − | <script src="https://2018.igem.org/Team:UPF_CRG_Barcelona/scripts?action=raw&ctype=text/javascript" charset="utf-8" type="text/javascript">
| |
| − | </script>
| |
| − | <script>
| |
| − | // Initialize Swiper
| |
| − | var swiper = new Swiper('.swiper-container', {
| |
| − | slidesPerView: 6,
| |
| − | slidesPerGroup: 1,
| |
| − | loop: true,
| |
| − | freeMode: true,
| |
| − | observer: true,
| |
| − | watchOverflow: true,
| |
| − | navigation: {
| |
| − | nextEl: '.swiper-button-next',
| |
| − | prevEl: '.swiper-button-prev',
| |
| − | },
| |
| − | keyboard: {
| |
| − | enabled: true,
| |
| − | },
| |
| − | watchOverflow: true,
| |
| − | freeModeSticky: true,
| |
| − | mousewheel: {
| |
| − | forceToAxis: true,
| |
| − | },
| |
| − | preventClicks: true,
| |
| − | iOSEdgeSwipeDetection: true,
| |
| − | observeParents: true,
| |
| − | breakpoints: {
| |
| − | 1024: {
| |
| − | slidesPerView: 5,
| |
| − | },
| |
| − | 768: {
| |
| − | slidesPerView: 4,
| |
| − | },
| |
| − | 640: {
| |
| − | slidesPerView: 3,
| |
| − | },
| |
| − | 500: {
| |
| − | slidesPerView: 2,
| |
| − | },
| |
| − | 320: {
| |
| − | slidesPerView: 1,
| |
| − | }
| |
| − | },
| |
| | }); | | }); |
| | | | |
| Line 2,008: |
Line 1,096: |
| | }) | | }) |
| | Plotly.relayout('results_biosensor5', { | | Plotly.relayout('results_biosensor5', { |
| − | width: Math.round(document.body.clientWidth * 0.7),
| |
| − | })
| |
| − | Plotly.relayout('results_biosensor6', {
| |
| | width: Math.round(document.body.clientWidth * 0.7), | | width: Math.round(document.body.clientWidth * 0.7), |
| | }) | | }) |
| | }; | | }; |
| − | </script>
| + | </script> |
| | </body> | | </body> |
| | | | |
| | </html> | | </html> |
