Difference between revisions of "Team:UPF CRG Barcelona/Improve"

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<p class="subapart1">Characterization</p>
 
<p class="subapart1">Characterization</p>
  
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<p>FOTOOOOO</p>
+
    <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,
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                                    color: "#36393d",
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                                },
 +
                            },
 +
                            yaxis: {
 +
                                zeroline: false,
 +
                                range: [0.99, 3],
 +
                                title: "Fold Change (FC/LB)",
 +
                                titlefont: {
 +
                                    family: "PT Sans",
 +
                                    size: 15,
 +
                                    color: "#36393d",
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                                },
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                                showticklabels: true,
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                                tickfont: {
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                                    family: "PT Sans",
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                                    color: "#36393d",
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                                },
 +
                            },
 +
                        };
 +
                        Plotly.newPlot('results_biosensor6', data, layout, {
 +
                            displayModeBar: false
 +
                        });
 +
                    </script>
  
 
<p>Our results showed an increased fold change after induction of the pAR promoter with different PA concentrations. Moreover, when compared with figure (x) we see a difference in the fold change induction. Suggesting that our PA dependent promoter responds to PA in a more on/off switch behavior. </p>
 
<p>Our results showed an increased fold change after induction of the pAR promoter with different PA concentrations. Moreover, when compared with figure (x) we see a difference in the fold change induction. Suggesting that our PA dependent promoter responds to PA in a more on/off switch behavior. </p>
 
<p>Taken together, our results suggest that pAR has a higher dynamic range than pFadBA, being a suitable candidate for a LCFA biosensor.</p>
 
<p>Taken together, our results suggest that pAR has a higher dynamic range than pFadBA, being a suitable candidate for a LCFA biosensor.</p>
  
−
<p class="references">[1]Feng Y, Cronan JE Jr: Crosstalk of Escherichia coli FadR with global regulators in expression of fatty acid transport genes. PLoS One 2012, 7:e46275.</p>
 
−
<p class="references">[2]NTU_Taida 2014 Wiki page.  https://2014.igem.org/Team:NTU_Taida </p>
 
−
 
−
<p class="references">[3] Zhang, F., Carothers, J. M., & Keasling, J. D. (2012). Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nature biotechnology, 30(4), 354.</p>
 
  
  
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       </div>
 
       </div>
  
 +
<p><b>Reference</b><p>
 +
<p class="references">[1]Feng Y, Cronan JE Jr: Crosstalk of Escherichia coli FadR with global regulators in expression of fatty acid transport genes. PLoS One 2012, 7:e46275.</p>
 +
<p class="references">[2]NTU_Taida 2014 Wiki page.  https://2014.igem.org/Team:NTU_Taida </p>
 +
 +
<p class="references">[3] Zhang, F., Carothers, J. M., & Keasling, J. D. (2012). Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nature biotechnology, 30(4), 354.</p>
 
     </section>
 
     </section>
  

Revision as of 17:15, 17 October 2018

Wiki

IMPROVED PARTS: Optimizing the dynamic range of the promoter

BBa_K2581011: Improved fatty acid acyl-CoA biosensor with medium RBS

The UPF_CRG_Barcelona iGEM team 2018 has create this part as an improved element from the existing fatty acid intracellular promoter pFadBA (BBa_K817002).

This biobrick consists in the assembly of a double terminator which allows forward and reverse terminator (BBa_B0014), our improved promoter based on the previous pFadBA DNA sequence (BBa_K2581013), a weak RBS (BBa_B0032) and a reporter gene, an engineered mutant of red fluorescent protein from Discosoma striata (BBa_E1010).

Introduction

pFadBA (BBa_K817002) promoter is a natural LCFA biosensor. It is the promoter of the endogenous E. coli fadB and fadA genes and contains FadR binding sequences [1]. FadR is the main transcriptional regulator of the beta oxidation pathway, as it is constitutively repressing the fad genes. The DNA-binding activity of FadR is antagonyzed by intracellular LCFA-AcylCoA, thus, in the presence of intracellular LCFA the promoter is derepressed allowing the expression of the fad genes.

Other iGEM teams have previously attempted to use it as a LCFA sensor, such as NTU_Taida 2014 [2]. However, their results showed a very high baseline expression of the reporter proteins coupled to the promoter. This did not allow them to see a significant rise in the signal after induction with LCFA.

Consequently, as pFadBA is a sensor with excessive leakage and a poor dynamic range our team tried to develop a better LFCA biosensor. Zhang et al. 2012 described a synthetic promoter with a higher dynamic range (pAR, BBa_K2581012), which we have characterized for the first time to avoid these levels of basality [3]. In short, this promoter contains an additional FadR binding sequence than the natural one.

In order to evaluate the responses of this promoter, we builded a circuit with pAR coupled to fluorescent reporter (BBa_E1010). Top10 bacteria (DH5-alpha) expressing the construct were induced with different concentrations of PA in LB media. Fluorescence and OD600nm was analyzed once it had reached the steady state(13-15h).

Characterization

Our results showed an increased fold change after induction of the pAR promoter with different PA concentrations. Moreover, when compared with figure (x) we see a difference in the fold change induction. Suggesting that our PA dependent promoter responds to PA in a more on/off switch behavior.

Taken together, our results suggest that pAR has a higher dynamic range than pFadBA, being a suitable candidate for a LCFA biosensor.

Reference

[1]Feng Y, Cronan JE Jr: Crosstalk of Escherichia coli FadR with global regulators in expression of fatty acid transport genes. PLoS One 2012, 7:e46275.

[2]NTU_Taida 2014 Wiki page. https://2014.igem.org/Team:NTU_Taida

[3] Zhang, F., Carothers, J. M., & Keasling, J. D. (2012). Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nature biotechnology, 30(4), 354.