Difference between revisions of "Part:BBa K2273000"
 
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<partinfo>BBa_K2273000 short</partinfo>
 
<partinfo>BBa_K2273000 short</partinfo>
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<html><figure><figure style="width:50%;float:right;"><img src="https://static.igem.org/mediawiki/2017/6/60/T--TU_Dresden--P_Communication_Figure1.jpeg" width="100%" class="zoom"><figcaption><b>Figure 1: The regulatory system for competence development in <i><b>Bacillus subtilis</b></i>.</b> The development of genetic competence is stimulated by quorum sensing due to the secretion of the ComX pheromone and its accumulation in the surrounding medium. (Modified from: <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/22511326">Jung <i>et al.</i></a>)</figcation></figure></figure></html>
 
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===Usage and Biology===
 
===Usage and Biology===
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<html><figure><p align="justify">This promoter is controlled by the well-studied regulatory system for competence development in <i>Bacillus subtilis</i> (Figure 1).
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<html>
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<i>B. subtilis</i> constantly secretes the ComX pheromone (<a href="https://parts.igem.org/Part:BBa_K2273003">BBa_K2273000</a>), a 9- to 10-amino acid oligopeptide, as a signalling molecule <b>(a)</b>. By rising cell-density, the ComX-concentration in the surrounding medium increases until it reaches a threshold and activates ComP, a membrane-spanning protein kinase <b>(b)</b>. This mechanism is called quorum sensing.
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<figure>
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The kinase reacts to the accumulation of ComX by phosphorylating the response regulator ComA <b>(c)</b> which then works as a transcription factor by binding to several promoters and enhancing their activity <b>(d)</b>.</p>
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<figure style="width:50%;float:right;"><img src="https://static.igem.org/mediawiki/2017/6/60/T--TU_Dresden--P_Communication_Figure1.jpeg" width="100%"/>
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<p align="justify">
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<figcaption><p align="justify"><b>Figure 1: The regulatory system for competence development in <i><b>Bacillus subtilis</b></i>.</b> The development of genetic competence is stimulated by quorum sensing due to the secretion of the ComX pheromone and its accumulation in the surrounding medium. (Modified from: <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/22511326">Jung <i>et al.</i>)</a></p></figcation>
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The most important promoter regulated by ComA and involved in competence development is the promoter of the <i>srfA</i> operon (P<sub><i>srfA</i></sub>). This operon contains not only genes for the production of the antibiotic surfactin <b>(e)</b> but also for ComS <b>(f)</b>, another small peptide that prevents the degradation of the autoregulated transcription factor ComK <b>(g)</b>. Since the promoter is not only dependent on quorum sensing but also on nutritional conditions, we evaluated the influence of different media (Figure 2). We tested full medium (LB) as well as minimal media (MNGE, MCSE).</p><figure style="padding-right:10%; padding-left:10%"><img style="width:80%" src="https://static.igem.org/mediawiki/parts/1/11/T--TU_Dresden--P_Communication_Psrf_Media.png"><figcaption><b>Figure 2: Growth (A) and promoter activity of P<sub><i><b>srfA</b></i></sub> (B) in different media.</b> Wild type (WT, pink) and <i>comX</i>-knockout strains (<i>ΔcomX</i>, dark blue) contain a P<sub><i>srfA</i></sub> <i>luxABCDE</i> fusion. </figcation></figure></figure>
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</figure>
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<p align="justify">This promoter is part of the well-studied regulatory system for competence development in <i>Bacillus subtilis</i> (Figure 1).
 +
<i>B. subtilis</i> constantly secretes the ComX pheromone (<a href="https://parts.igem.org/Part:BBa_K2273003">BBa_K2273003</a>), a 9- to 10-amino acid oligopeptide, as a signalling molecule <b>(a)</b>. By rising cell-density, the ComX-concentration in the surrounding medium increases until it reaches a threshold and activates ComP, a membrane-spanning protein kinase <b>(b)</b>. This mechanism is called quorum sensing.
 +
The kinase reacts to the accumulation of ComX by phosphorylating the response regulator ComA <b>(c)</b> which then works as a transcription factor by binding to several promoters and enhancing their activity <b>(d)</b>.
 +
<a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/12576575">(Hamoen <i>et al.</i>)</a><br>
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The most important promoter regulated by ComA and involved in competence development is the promoter of the <i>srfA</i> operon (P<sub><i>srfA</i></sub>). This operon contains not only genes for the production of the antibiotic surfactin <b>(e)</b> but also for ComS <b>(f)</b>, another small peptide that prevents the degradation of the autoregulated transcription factor ComK <b>(g)</b>. <br>Since the promoter is not only dependent on quorum sensing but also on nutritional conditions, we evaluated the influence of different media (Figure 2). We tested full medium (LB) as well as minimal media (MNGE, MCSE).</p>
  
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<p align="justify">Furthermore, we studied the influence of ComX-overproduction on the promoter activity by integrating an inducible copy of <i>comX<i>.</p>
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</figure>
  
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<figure><figure style="padding-left:30%; padding-right:30%"><img src="https://static.igem.org/mediawiki/parts/a/a3/T--TU_Dresden--P_Communication_Psrf_Induction.png" width="100%" class="zoom"><figcaption><b>Growth (A) and promoter activity of P<sub><i><b>srfA</b></i></sub> (B) in dependence of xylose-induction.</b> Wild type (WT, pink and light blue) and <i>comX</i>-knockout strain (<i>ΔcomX</i>, black and dark blue) contain a P<sub><i>srfA</i></sub>- <i>luxABCDE</i>fusion. After 1 hour of incubation (dashed line) the cultures were induced with 1% xylose (light blue and black) or with distilled water (dH2O, pink and dark blue).</figcation></figure></figure>
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<figure style="padding-right:15%; padding-left:15%;">
  
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<b>The promoter was created and used by <a target="_blank" href="http://2017.igem.org/Team:TU_Dresden/Project/Communication">TU Dresden iGEM team</a>
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<img src="https://static.igem.org/mediawiki/parts/1/11/T--TU_Dresden--P_Communication_Psrf_Media.png" width="100%"/>
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<figcaption><p align="justify"><b>Figure 2: Growth (A) and promoter activity of P<sub><i><b>srfA</b></i></sub> (B) in different media.</b> Wild type (WT, pink) and <i>comX</i>-knockout strains (Δ<i>comX</i>, dark blue) contain a P<sub><i>srfA</i></sub>-<i>luxABCDE</i> fusion.</p></figcation>
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</figure>
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<p align="justify">Furthermore, we studied the influence of ComX-overproduction on the promoter activity by integrating an inducible copy of <i>comX</i>.</p>
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 +
<figure style="padding-left:35%; padding-right:35%;"><img src="https://static.igem.org/mediawiki/parts/a/a3/T--TU_Dresden--P_Communication_Psrf_Induction.png" width="100%"/><figcaption><p align="justify"><b>Growth (A) and promoter activity of P<sub><i><b>srfA</b></i></sub> (B) in dependence of xylose-induction.</b> Wild type (WT, pink and light blue) and <i>comX</i>-knockout strain (Δ<i>comX</i>, black and dark blue) contain a P<sub><i>srfA</i></sub>-<i>luxABCDE</i> fusion. After 1 hour of incubation (dashed line) the cultures were induced with 1% xylose (light blue and black) or with distilled water (dH2O, pink and dark blue).</p></figcation>
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</figure>
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<p align="justify">
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<b>The promoter was created and used by <a target="_blank" href="http://2017.igem.org/Team:TU_Dresden/Project/Communication">TU_Dresden 2017</a>.</b>  All assays have been conducted with <i>B. subtilis</i> W168 carrying the promoter fused to the <i>lux</i> operon.
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Other promoters that haven been evaluated regarding their ComX-dependent activity:
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<table>
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<tr><td>P<sub><i>srfA</i></sub></td><td><a href="https://parts.igem.org/Part:BBa_K2273000">BBa_K2273000</a></td></tr>
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<tr><td>P<sub><i>rapA</i></sub></td><td><a href="https://parts.igem.org/Part:BBa_K2273001">BBa_K2273001</a></td></tr>
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<tr><td>P<sub><i>rapF</i></sub></td><td><a href="https://parts.igem.org/Part:BBa_K2273002">BBa_K2273002</a></td></tr>
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<tr><td>P<sub><i>comG</i></sub></td><td><a href="https://parts.igem.org/Part:BBa_K2273012">BBa_K2273012</a></td></tr>
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<tr><td>P<sub><i>comK</i></sub> mut</td><td><a href="https://parts.igem.org/Part:BBa_K2273013">BBa_K2273013</a></td></tr>
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</table>
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<br></p>
 
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</html>
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2273000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2273000 SequenceAndFeatures</partinfo>
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Latest revision as of 14:21, 31 October 2017


PsrfA: a ComA-dependent promoter of Bacillus subtilis

Usage and Biology

Figure 1: The regulatory system for competence development in Bacillus subtilis. The development of genetic competence is stimulated by quorum sensing due to the secretion of the ComX pheromone and its accumulation in the surrounding medium. (Modified from: Jung et al.)

This promoter is part of the well-studied regulatory system for competence development in Bacillus subtilis (Figure 1). B. subtilis constantly secretes the ComX pheromone (BBa_K2273003), a 9- to 10-amino acid oligopeptide, as a signalling molecule (a). By rising cell-density, the ComX-concentration in the surrounding medium increases until it reaches a threshold and activates ComP, a membrane-spanning protein kinase (b). This mechanism is called quorum sensing. The kinase reacts to the accumulation of ComX by phosphorylating the response regulator ComA (c) which then works as a transcription factor by binding to several promoters and enhancing their activity (d). (Hamoen et al.)
The most important promoter regulated by ComA and involved in competence development is the promoter of the srfA operon (PsrfA). This operon contains not only genes for the production of the antibiotic surfactin (e) but also for ComS (f), another small peptide that prevents the degradation of the autoregulated transcription factor ComK (g).
Since the promoter is not only dependent on quorum sensing but also on nutritional conditions, we evaluated the influence of different media (Figure 2). We tested full medium (LB) as well as minimal media (MNGE, MCSE).

Figure 2: Growth (A) and promoter activity of PsrfA (B) in different media. Wild type (WT, pink) and comX-knockout strains (ΔcomX, dark blue) contain a PsrfA-luxABCDE fusion.

Furthermore, we studied the influence of ComX-overproduction on the promoter activity by integrating an inducible copy of comX.

Growth (A) and promoter activity of PsrfA (B) in dependence of xylose-induction. Wild type (WT, pink and light blue) and comX-knockout strain (ΔcomX, black and dark blue) contain a PsrfA-luxABCDE fusion. After 1 hour of incubation (dashed line) the cultures were induced with 1% xylose (light blue and black) or with distilled water (dH2O, pink and dark blue).

The promoter was created and used by TU_Dresden 2017. All assays have been conducted with B. subtilis W168 carrying the promoter fused to the lux operon. Other promoters that haven been evaluated regarding their ComX-dependent activity:

PsrfABBa_K2273000
PrapABBa_K2273001
PrapFBBa_K2273002
PcomGBBa_K2273012
PcomK mutBBa_K2273013

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]