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

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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
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            <p>Basic Parts</p>
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            <p>Improved Parts</p>
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  <main>
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<h1>Improve</h1>
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<p>For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Registry. Please include a link to your improved part on this page.</p>
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<h3>Gold Medal Criterion #2</h3>
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    <section class="">
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<p><b>Standard Tracks:</b> Create a new part that has a functional improvement upon an existing BioBrick part. The sequences of the new and existing parts must be different. You must perform experiments with both parts to demonstrate this improvement.  Document the experimental characterization on the Part's Main Page on the Registry for both the existing and new parts. Both the new and existing Main Page of each Part’s Registry entry must reference each other. Submit a sample of the new part to the Registry.
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The existing part must NOT be from your 2018 part number range and must be different from the part documented in bronze #4.
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      <div>
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        <p class="page-title">IMPROVED PARTS: Optimizing the dynamic range of the promoter</p>
  
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<br><br>
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        <p><b>BBa_K2581011: Improved fatty acid acyl-CoA biosensor with medium RBS</b></p>
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<b>Special Tracks:</b> Improve the function of an existing iGEM project (that your current team did not originally create) and display your achievement on your wiki.</p>
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        <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).</p>
 +
        <p>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 <i>Discosoma striata</i>
 +
          (BBa_E1010).
 +
        </p>
 +
    <div class="spacer"></div>
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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>
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    <div class="spacer"></div>
  
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<p class="subapart1">Introduction</p>
 +
   
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        <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>
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<p class="subapart1">Characterization</p>
  
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</div>
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                    <script>
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                        var pFadBA = {
 +
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                            y: [1, 1.131746482, 1.542923974],
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                        var pAR = {
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                            x: ['LB', 'LB 0.4 mM PA', 'LB 1 mM PA'],
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                            y: [1, 2.848309281, 3.19905373],
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                        var data = [pFadBA, pAR];
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                            title: "Comparison of Fold Change between pFadBA and pAR",
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<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>
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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.