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             <!--class is-active for showing active menu-->
 
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       <div>
 
       <div>
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         <p class="page-title">IMPROVED PARTS</p>
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         <p class="page-title">IMPROVED PARTS: Optimizing the dynamic range of the promoter</p>
  
−
         <p><b>BBa_K2581011: T14_pAR_32_RFP or Improved fatty acid acyl-CoA biosensor with medium RBS</b></p>
+
         <p><b>BBa_K2581011: Improved fatty acid acyl-CoA biosensor with medium RBS</b></p>
−
         <p>The UPF_CRG_Barcelona iGEM team 2018 has create this part as an improved element from the existing fatty
+
         <p>The UPF_CRG_Barcelona iGEM team 2018 has created this part as an improved element from the existing fatty
−
           acid intracellular promoter pFadBA (BBa_K817002). It act as an intracellular biosensor of fatty acid acyl-coA
+
           acid intracellular promoter pFadBA (BBa_K817002).</p>
−
          but in this case the high basality activity of the promoter has been reduced. </p>
+
         <p>This biobrick consists in the assembly of a double terminator which allows for forward and reverse termination
−
         <p>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_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 <i>Discosoma striata</i>
 
           (BBa_B0032) and a reporter gene, an engineered mutant of red fluorescent protein from <i>Discosoma striata</i>
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         <center><img src="https://static.igem.org/mediawiki/2018/e/ec/T--UPF_CRG_Barcelona--goldenpart32.svg" style="width: 50%;"></center>
 
         <center><img src="https://static.igem.org/mediawiki/2018/e/ec/T--UPF_CRG_Barcelona--goldenpart32.svg" style="width: 50%;"></center>
 
     <div class="spacer"></div>
 
     <div class="spacer"></div>
−
        <p><b>BBa_K2581012: T14_pAR_34_RFP or Improved fatty acid acyl-CoA biosensor with strong RBS</b></p>
 
−
        <p>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). It act as an intracellular biosensor of fatty acid acyl-coA
 
−
          but in this case the high basality activity of the promoter has been reduced.</p>
 
−
        <p>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), an strong RBS
 
−
          (BBa_B0034) and a reporter gene, an engineered mutant of red fluorescent protein from <i>Discosoma striata</i>
 
−
          (BBa_E1010). </p>
 
−
    <div class="spacer"></div>
 
−
        <center><img src="https://static.igem.org/mediawiki/2018/5/5c/T--UPF_CRG_Barcelona--goldenpart34.svg" style="width: 50%;"></center>
 
−
    <div class="spacer"></div>
 
−
      </div>
 
  
 +
<p class="subapart1">Introduction</p>
 +
   
 +
        <p>pFadBA (BBa_K817002) promoter is a natural LCFA biosensor. It is the promoter of the endogenous <i>E. coli</i> 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.</p>
 +
        <p>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.</p>
 +
<p>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.</p>
 +
<p>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).</p>
 +
<p class="subapart1">Characterization</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,
 +
                                    color: "#36393d",
 +
                                },
 +
                            },
 +
                            yaxis: {
 +
                                zeroline: false,
 +
                                range: [0.99, 3],
 +
                                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>
 +
 +
<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><b>References
 +
</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>
−
    <div class="spacer"></div>
 
  
 
   </main>
 
   </main>
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       },
 
       },
 
     });
 
     });
 +
    window.onresize = function () {
 +
        Plotly.relayout('results_biosensor6', {
 +
            width: Math.round(document.body.clientWidth * 0.7),
 +
        })
 +
    };
 
   </script>
 
   </script>
 
</body>
 
</body>

Latest revision as of 00:42, 18 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 created 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 for forward and reverse termination (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.

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.

[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.