Difference between revisions of "Part:BBa K104001"

Revision as of 04:14, 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

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.

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.

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Fig2 SDS-PAGE to confirm the GFP expression

However, we found a heavy leak of the promoter when the plasmid was transformed in BL21

<center>Fig3 Phenotype of GFP-expressing colonies of BL21