Difference between revisions of "Part:BBa K2273002"
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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%"/> | <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%"/> | ||
| − | <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> | + | <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> |
</figure> | </figure> | ||
<p align="justify">This promoter is under the control of the well-studied regulatory system for competence development in <i>Bacillus subtilis</i> (Figure 1). | <p align="justify">This promoter is under the control 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. | <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>.</br> | + | 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> |
| − | According to literature the promoter P<sub><i> | + | According to literature the promoter P<sub><i>rapF</i></sub> is regulated by ComA. Its sequence contains a canonical and non-canonical binding site of ComA (<a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/?term=The%20quorum-sensing%20regulator%20ComA%20from%20Bacillus%20subtilis%20activates%20transcription%20using%20topologically%20distinct%20DNA%20motifs">Wolf <i>et al.</i>)</a>.<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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<figure style="padding-right:15%; padding-left:15%;"> | <figure style="padding-right:15%; padding-left:15%;"> | ||
| − | <img src="https://static.igem.org/mediawiki/parts/ | + | <img src="https://static.igem.org/mediawiki/parts/a/a2/T--TU_Dresden--P_Communication_PrapF_Media.png" width="100%"/> |
| − | <figcaption><p align="justify"><b>Figure 2: Growth (A) and promoter activity of P<sub><i><b> | + | <figcaption><p align="justify"><b>Figure 2: Growth (A) and promoter activity of P<sub><i><b>rapF</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>rapF</i></sub>-<i>luxABCDE</i> fusion.</p></figcation> |
</figure> | </figure> | ||
<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> | <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> | ||
| − | <figure style="padding-left:35%; padding-right:35%;"><img src="https://static.igem.org/mediawiki/parts/ | + | <figure style="padding-left:35%; padding-right:35%;"><img src="https://static.igem.org/mediawiki/parts/0/04/T--TU_Dresden--P_Communication_PrapF_Induction.png" width="100%"/><figcaption><p align="justify"><b>Growth (A) and promoter activity of P<sub><i><b>rapF</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>rapF</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> |
</figure> | </figure> | ||
<p align="justify"> | <p align="justify"> | ||
Latest revision as of 16:16, 31 October 2017
PrapF: 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 under the control 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.)
According to literature the promoter PrapF is regulated by ComA. Its sequence contains a canonical and non-canonical binding site of ComA (Wolf et al.). Figure 2: Growth (A) and promoter activity of PrapF (B) in different media. Wild type (WT, pink) and comX-knockout strains (ΔcomX, dark blue) contain a PrapF-luxABCDE fusion.
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).
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 PrapF (B) in dependence of xylose-induction. Wild type (WT, pink and light blue) and comX-knockout strain (ΔcomX, black and dark blue) contain a PrapF-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:
| PsrfA | BBa_K2273000 |
| PrapA | BBa_K2273001 |
| PrapF | BBa_K2273002 |
| PcomG | BBa_K2273012 |
| PcomK mut | BBa_K2273013 |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]