Difference between revisions of "Part:BBa K1031221"
 
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<partinfo>BBa_K1031221 short</partinfo>
 
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<p>For detailed information concerning DmpR and <i>Po</i> promoter, please visit <a href="http://2013.igem.org/Team:Peking/Project/BioSensors/DmpR">2013 Peking iGEM Biosensor DmpR</a></p>
  
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Po promoter which is activated by DmpR, is σ-54 dependent. It is composed of three regions. The two inverted UAS sites are responsible for binding of DmpR transcriptional factor. The two IHF binding sites allowing IHF to participate enhance transcription efficiency. -24 and -12 region interact with sigma-54 factor of RNA polymerase, enabling the formation of open complex. ('''Fig 1''')
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<i>''Po''</i> promoter which is activated by DmpR, is σ-54 dependent. It is composed of three elements. The UAS site containing two inverted binding regions is responsible for interacting with DmpR transcriptional factor. The two IHF binding sites allowing IHF to participate, enhance transcription efficiency. -24 and -12 region interact with σ-54 factor of RNA polymerase, enabling the formation of open complex. ('''Fig 1''')
  
 
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<p style="text-align:center"><b>Fig 1</b> <i>Po</i> promoter structure. The UAS of this promoter marked in green is combined of two parts in contrast direction to which DmpR binds. The box with yellow background represents IHF binding sites. The box with pink background represents σ54 binding site with -24 region and -12 region marked in red. The G with right angle represents +1 site.
 
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'''Fig 1'''  Po promoter structure. The UAS of this promoter marked in green is combined of two parts in contrast direction to which DmpR binds. The box with yellow background represents IHF binding sites. The box with pink background represents σ54 binding site with -24 region and -12 region marked in red. The G with right angle represents +1 site.
 
  
  
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We constructed a library of RBS (Ribosome Binding Site) of different strength for tuning the expression of reporter sfGFP.  
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We constructed a library of RBSs (Ribosome Binding Sites) of different strength for tuning the expression of reporter sfGFP (super folder GFP).  
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K1031221 is a reporter circuit composed of three elements, the inducible promoter ''Po''for DmpR, RBS B0031[https://parts.igem.org/Part:BBa_B0031], and reporter gene sfGFP.  ('''Fig 2''')  
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K1031221 is a reporter circuit composed of three elements, the inducible promoter ''Po''for DmpR, RBS <html><a href="https://parts.igem.org/Part:BBa_B0031">B0031</a></html>, and reporter gene sfGFP with terminator<html><a href="https://parts.igem.org/Part:BBa_B0015">B0015</a></html>.  ('''Fig 2''')  
  
 
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<p style="text-align:center"><b>Fig 2</b> Construction of reporter circuit. The orange arrow represents <i>Po</i> promoter for DmpR. The green oval stands for RBS B0031. sfGFP coding sequence is shown with dark blue, while terminator B0015 is in dark red.
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'''Fig 2''' Construction of reporter circuit. The orange arrow represents ''Po'' promoter for DmpR. The green oval stands for RBS B0031. sfGFP coding sequence is shown with dark blue, while terminator B0015[https://parts.igem.org/Part:BBa_B0015] is in dark red.
 
  
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The performances of reporter circuits adopting B0031 and B0032 in collocation with ''Pc''-DmpR were tested('''Fig 3'''). The reporter circuit consists of '' Po'' promoter, RBS B0031, and reporter gene sfGFP. The dashed box refers to data for reporter circuit adopting RBS B0031.
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The performance of each reporter circuit adopting B0031, B0032, B0033 and B0034 in collocation with ''Po''/DmpR circuit was tested.('''Fig 3''')
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<img src="https://static.igem.org/mediawiki/igem.org/thumb/8/8f/Peking2013_part_31-Po_DmpR.png/800px-Peking2013_part_31-Po_DmpR.png" style="width:500px; margin-left:210px" />
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<p style="text-align:center"><b>Fig 3</b> Fluorescence intensity of DmpR biosensor at the absence and presence of inducer following <a href="http://2013.igem.org/Team:Peking/Team/Notebook/Protocols">Test Protocol 1</a>. The line of light blue represents fluorescence background, and the line of dark blue stands for fluorescence intensity when exposed to phenol. The dashed box refers to data for biosensor <i>Po</i>/DmpR adopting RBS B0031.
 
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'''Fig 3'''Induction ratio of DmpR when exposed to a series of concentration of ***. The reporter circuit includes'' Po''-RBS-sfGFP. Three lines represent sfGFP controlled by different RBS. Fluorescence intensity of sfGFP is detected and calculated to plot induction ratio
 
  
  

Latest revision as of 02:51, 28 September 2013

Po-B0031-sfGFP-Terminator (DmpR)

For detailed information concerning DmpR and Po promoter, please visit 2013 Peking iGEM Biosensor DmpR


Structure

Po promoter which is activated by DmpR, is σ-54 dependent. It is composed of three elements. The UAS site containing two inverted binding regions is responsible for interacting with DmpR transcriptional factor. The two IHF binding sites allowing IHF to participate, enhance transcription efficiency. -24 and -12 region interact with σ-54 factor of RNA polymerase, enabling the formation of open complex. (Fig 1)

Fig 1 Po promoter structure. The UAS of this promoter marked in green is combined of two parts in contrast direction to which DmpR binds. The box with yellow background represents IHF binding sites. The box with pink background represents σ54 binding site with -24 region and -12 region marked in red. The G with right angle represents +1 site.


Sequence and Features

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 212


Construction and data

We constructed a library of RBSs (Ribosome Binding Sites) of different strength for tuning the expression of reporter sfGFP (super folder GFP). K1031221 is a reporter circuit composed of three elements, the inducible promoter Pofor DmpR, RBS B0031, and reporter gene sfGFP with terminatorB0015. (Fig 2)

Fig 2 Construction of reporter circuit. The orange arrow represents Po promoter for DmpR. The green oval stands for RBS B0031. sfGFP coding sequence is shown with dark blue, while terminator B0015 is in dark red.


The performances of reporter circuits adopting B0031 and B0032 in collocation with Pc-DmpR were tested(Fig 3). The reporter circuit consists of Po promoter, RBS B0031, and reporter gene sfGFP. The dashed box refers to data for reporter circuit adopting RBS B0031.

Fig 3 Fluorescence intensity of DmpR biosensor at the absence and presence of inducer following Test Protocol 1. The line of light blue represents fluorescence background, and the line of dark blue stands for fluorescence intensity when exposed to phenol. The dashed box refers to data for biosensor Po/DmpR adopting RBS B0031.