Difference between revisions of "Part:BBa K2273013"

 
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<partinfo>BBa_K2273013 short</partinfo>
 
<partinfo>BBa_K2273013 short</partinfo>
  
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===Usage and Biology===
 
===Usage and Biology===
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<figure>
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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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<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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</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).
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<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.
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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>.
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<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>, <a href="https://parts.igem.org/Part:BBa_K2273000">BBa_K2273000</a>). 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>. ComK activates expression of more than 100 genes, including <i>comG</i>, that is part of the transformation machinery <b>(h)</b> <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/16091051">(Comella <i>et al.</i>,</a><a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/14762007">Susanna <i>et al.</i>)</a>. Consequently, <i>B. subtilis</i> can take up DNA from the environment either as direct nutrient source or incorporate the DNA via homologues recombination into its own genome.
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<br>Since the competence development 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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</figure>
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<figure style="padding-right:15%; padding-left:15%;">
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<img src="https://parts.igem.org/File:T--TU_Dresden--P_Communication_PcomK_mut_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>comK</b></i></sub> mut (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>comK</i></sub> mut-<i>luxABCDE</i> fusion.</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>
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2273013 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2273013 SequenceAndFeatures</partinfo>

Revision as of 15:42, 31 October 2017


PcomK (mutated): a ComK-dependent promoter

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, BBa_K2273000). 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). ComK activates expression of more than 100 genes, including comG, that is part of the transformation machinery (h) (Comella et al.,Susanna et al.). Consequently, B. subtilis can take up DNA from the environment either as direct nutrient source or incorporate the DNA via homologues recombination into its own genome.
Since the competence development 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 PcomK mut (B) in different media. Wild type (WT, pink) and comX-knockout strains (ΔcomX, dark blue) contain a PcomK mut-luxABCDE fusion.

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]