Difference between revisions of "Part:BBa K3071013"
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<partinfo>BBa_K3071013 short</partinfo> | <partinfo>BBa_K3071013 short</partinfo> | ||
===Description=== | ===Description=== | ||
| − | This is an regulatory element that originated from <i>Escherichia coli</i> K12 strain. This basic part is used to construct our composite part ([https://parts.igem.org/Part:BBa_K3071014 BBa_K3071014]). Our rt-qPCR data show that this | + | This is an regulatory element that originated from <i>Escherichia coli</i> K12 strain. This basic part is used to construct our composite part ([https://parts.igem.org/Part:BBa_K3071014 BBa_K3071014]). Our rt-qPCR data show that this promoter is able to initiate the transcription in form of synthetic construct. |
===Biology=== | ===Biology=== | ||
<center>[[File:T--Hong_Kong-CUHK--pspA_promotor_structure.png]]</center> | <center>[[File:T--Hong_Kong-CUHK--pspA_promotor_structure.png]]</center> | ||
| − | <center><u> Figure 1 Basic properties of pspA | + | <center><u> Figure 1 Basic properties of pspA promoter</u></center> |
| − | pspA | + | pspA promoter is a sigma-54 (σ-54) regulated activator dependent promoter. In the orginal pspA promoter upstream region, it contains σ-54 consensus sequence 5' of the start contains a GG doublet as -24 and a consensus GG doublet at -12, the high-affinity IHF site (-25 to -60), as well as the UAS sites (UAS I: -89 to -107; UAS II:-111 to -129). |
===Usage=== | ===Usage=== | ||
<center>[[File:T--Hong_Kong-CUHK--sigma_54_activation.png]]</center> | <center>[[File:T--Hong_Kong-CUHK--sigma_54_activation.png]]</center> | ||
| − | <center><u>Figure 2 The transcription initiation mechanism of the sigma-54 | + | <center><u>Figure 2 The transcription initiation mechanism of the sigma-54 promoters [http://www.cchem.berkeley.edu/wemmer/research/sigma54.html]</u></center> |
<br> | <br> | ||
| − | sigm54-RNA holoenzyme (σ-54 RNAP) forms an inactive transcriptional initiation complex on this | + | sigm54-RNA holoenzyme (σ-54 RNAP) forms an inactive transcriptional initiation complex on this promoter, which can be activated in E. coli by the bacterial enhancer-binding protein PspF (<html><a href="https://parts.igem.org/Part:BBa_K3071006">BBa_K3071006</a></html>). PspF functions by binding to the upstream activation sequences (UAS) near the promoter and contacting the promoter-bound σ-54 RNAP via DNA looping stabilized by the binding of integration host factor (IHF). Previous research has demonstrated the property of pspF-dependent and enhancer-specific transcription activation of pspA promoter. |
===Characterization=== | ===Characterization=== | ||
<center>[[file:T--Hong_Kong-CUHK--whole_design.png]]</center> | <center>[[file:T--Hong_Kong-CUHK--whole_design.png]]</center> | ||
| − | <center><u>Figure | + | <center><u>Figure 3 illustration of our synthetic biological system upon DSF activation</u></center> |
<br> | <br> | ||
| − | The UAS sites of this | + | The UAS sites of this promoter are replaced by CBS I(<html><a href="https://parts.igem.org/Part:BBa_K3071011">BBa_K3071011</a></html>) and CBSII (<html><a href="https://parts.igem.org/Part:BBa_K3071012">BBa_K3071012</a></html>) in our own synthetic system to construct CBS I & II-regulated pspA promoter (<html><a href="https://parts.igem.org/Part:BBa_K3071014">BBa_K3071014</a></html>). |
<center>[[file:T--Hong_Kong-CUHK--DSF_assay.png]]</center> | <center>[[file:T--Hong_Kong-CUHK--DSF_assay.png]]</center> | ||
| − | <center><u>Figure | + | <center><u>Figure 4 rt-qPCR data DSF treatment on the eforRED mRNA expression level</u></center> |
<br> | <br> | ||
The rt-qPCR data upon activation by DSF indicate a significant change in gene expression, suggest the related composite part is functional. | The rt-qPCR data upon activation by DSF indicate a significant change in gene expression, suggest the related composite part is functional. | ||
| + | |||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
Latest revision as of 08:07, 21 October 2019
PspA promoter
Description
This is an regulatory element that originated from Escherichia coli K12 strain. This basic part is used to construct our composite part (BBa_K3071014). Our rt-qPCR data show that this promoter is able to initiate the transcription in form of synthetic construct.
Biology
pspA promoter is a sigma-54 (σ-54) regulated activator dependent promoter. In the orginal pspA promoter upstream region, it contains σ-54 consensus sequence 5' of the start contains a GG doublet as -24 and a consensus GG doublet at -12, the high-affinity IHF site (-25 to -60), as well as the UAS sites (UAS I: -89 to -107; UAS II:-111 to -129).
Usage
sigm54-RNA holoenzyme (σ-54 RNAP) forms an inactive transcriptional initiation complex on this promoter, which can be activated in E. coli by the bacterial enhancer-binding protein PspF (BBa_K3071006). PspF functions by binding to the upstream activation sequences (UAS) near the promoter and contacting the promoter-bound σ-54 RNAP via DNA looping stabilized by the binding of integration host factor (IHF). Previous research has demonstrated the property of pspF-dependent and enhancer-specific transcription activation of pspA promoter.
Characterization
The UAS sites of this promoter are replaced by CBS I(BBa_K3071011) and CBSII (BBa_K3071012) in our own synthetic system to construct CBS I & II-regulated pspA promoter (BBa_K3071014).
The rt-qPCR data upon activation by DSF indicate a significant change in gene expression, suggest the related composite part is functional.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 12
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]