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

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        <p><b>Introduction</b></p>
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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>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>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>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>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><b>Characterization</b></p>
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<p class="subapart1">Characterization</p>
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<p>FOTOOOOO</p>
 
<p>FOTOOOOO</p>
  

Revision as of 15:21, 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

FOTOOOOO

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.

[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.