Difference between revisions of "Part:BBa K104001"
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<partinfo>BBa_K104001 short</partinfo>
 
<partinfo>BBa_K104001 short</partinfo>
  
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This part is for use in a Bacillus subtilis chassis. It encodes a two-component system that can be used to sense the level of a small lantibiotic peptide called subtilin. It belongs to the signalling series of parts (it is a receiver) and can be used to sense quorum sensing peptides from Gram positive organisms. In the Newcastle 2008 iGEM entry it was used a 'proof of concept' brick to test whether a quorum sensing peptide produced by one species or stain of bacteria could be sensed by a different strain or species.  
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This part is for use in a ''Bacillus subtilis'' chassis. It encodes a two-component system that can be used to sense the level of a small antibiotic peptide called subtilin. It belongs to the signalling series of parts (it is a receiver) and can be used to sense quorum sensing peptides from Gram positive organisms. In the [http://2008.igem.org/Team:Newcastle_University Newcastle 2008 iGEM entry] it was used a 'proof of concept' brick to test whether a quorum sensing peptide produced by one species or stain of bacteria could be sensed by a different strain or species.  
  
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The part contains two orfs arranged in an operon, SpaK and SpaR, that are driven by their native RBSs and the promoter PspaRK. Downstream of the SpaRK operon is terminator followed by a RBS and a promoter PspaS.
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The part contains two ORFs arranged in an operon, SpaK and SpaR, that are driven by their native RBSs and the promoter PspaRK. Downstream of the SpaRK operon is terminator followed by a RBS and a promoter PspaS.
  
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In response to subtilin, the sensor kinase SpaK will phosphorylate, and hence activate, the response regulator SpaR. The phosphorylated SpaR protein activates the PspaS promoter driving the expression of any downstream genes. In this brick there are no ORF's downstream of PspaS but the brick has been tested with gfp and mcherry by the Newcastle 2008 team.
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In response to subtilin, the sensor kinase SpaK will phosphorylate, and hence activate, the response regulator SpaR. The phosphorylated SpaR protein activates the PspaS promoter driving the expression of any downstream genes. In this part there are no ORFs downstream of PspaS but the part has been tested with GFP and mCherry by the Newcastle 2008 team.
  
 
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<partinfo>BBa_K104001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K104001 SequenceAndFeatures</partinfo>
  
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==User Reviews==
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Liaoliao_Gao<br>
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In 2022, ZJU-China iGEM team has characterized the output of this part in a novel chassis E. coli BL21(DE3) and a Bacillus subtilis strain WB600. The result was documented in the experience page and the main page of  (<partinfo>BBa_K104001</partinfo>).<br>
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===Contribution from iGEM2022 ZJU-China===
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<p>For signal module, we used GFP as the reporter gene to show the trigger of subtilisin by observing the bacterial colony’s phenotype and running SDS-PAGE.Therefore, we constructed PHY-Signal-GFP plasmid. Then we inoculated WB600 transformed by PHY-signaling-GFP plasmid on the media with or without 0.1mg/ml subtilisin.It’s quite clear that GFP was induced by 100μg/ml subtilisin, even though the growth of WB600 was moderately blocked by subtilisin.</p>
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<div align="center">[[File:GLL-3.png|600px]]</div>
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<center>Fig1 Phenotype of GFP-expressing colonies campared with the control group
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</center>
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<p>In order to guarantee the expression of GFP, we ran the SDS-PAGE later. Compared with the control group, the WB600 cultured with 0.1mg/ml subtilisin extended significantly strengthened expression of GFP.</p>
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<div align="center">[[File:GLL-4.png|600px]]</div>
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<center>Fig2  SDS-PAGE to confirm the GFP expression
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<p>However, we found a heavy leak of the promoter when the plasmid was transformed in BL21 with no subtilisin in the medium.</p>
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<div align="center">[[File:GLL-F7.jpeg|600px]]</div>
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<center>Fig3  Phenotype of GFP-expressing colonies of BL21(leak)
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K104001 parameters</partinfo>
 
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Latest revision as of 04:17, 26 September 2022

Sensor for small peptide Subtilin

This part is for use in a Bacillus subtilis chassis. It encodes a two-component system that can be used to sense the level of a small antibiotic peptide called subtilin. It belongs to the signalling series of parts (it is a receiver) and can be used to sense quorum sensing peptides from Gram positive organisms. In the [http://2008.igem.org/Team:Newcastle_University Newcastle 2008 iGEM entry] it was used a 'proof of concept' brick to test whether a quorum sensing peptide produced by one species or stain of bacteria could be sensed by a different strain or species.

The part contains two ORFs arranged in an operon, SpaK and SpaR, that are driven by their native RBSs and the promoter PspaRK. Downstream of the SpaRK operon is terminator followed by a RBS and a promoter PspaS.

In response to subtilin, the sensor kinase SpaK will phosphorylate, and hence activate, the response regulator SpaR. The phosphorylated SpaR protein activates the PspaS promoter driving the expression of any downstream genes. In this part there are no ORFs downstream of PspaS but the part has been tested with GFP and mCherry by the Newcastle 2008 team.

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]

User Reviews

Liaoliao_Gao
In 2022, ZJU-China iGEM team has characterized the output of this part in a novel chassis E. coli BL21(DE3) and a Bacillus subtilis strain WB600. The result was documented in the experience page and the main page of (BBa_K104001).

Contribution from iGEM2022 ZJU-China

For signal module, we used GFP as the reporter gene to show the trigger of subtilisin by observing the bacterial colony’s phenotype and running SDS-PAGE.Therefore, we constructed PHY-Signal-GFP plasmid. Then we inoculated WB600 transformed by PHY-signaling-GFP plasmid on the media with or without 0.1mg/ml subtilisin.It’s quite clear that GFP was induced by 100μg/ml subtilisin, even though the growth of WB600 was moderately blocked by subtilisin.

GLL-3.png
Fig1 Phenotype of GFP-expressing colonies campared with the control group

In order to guarantee the expression of GFP, we ran the SDS-PAGE later. Compared with the control group, the WB600 cultured with 0.1mg/ml subtilisin extended significantly strengthened expression of GFP.

GLL-4.png
Fig2 SDS-PAGE to confirm the GFP expression

However, we found a heavy leak of the promoter when the plasmid was transformed in BL21 with no subtilisin in the medium.

GLL-F7.jpeg

<center>Fig3 Phenotype of GFP-expressing colonies of BL21(leak)