Difference between revisions of "Part:BBa K104001:Experience"

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An ideal addition would be a resistance brick. Without the resistance genes, subtilin will kill Bacillus subtilis 168, causing cell lysis at high concentrations.
 
An ideal addition would be a resistance brick. Without the resistance genes, subtilin will kill Bacillus subtilis 168, causing cell lysis at high concentrations.
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See the [http://2008.igem.org/Team:Newcastle_University Newcastle] 2007 wiki for more details .
  
 
===User Reviews===
 
===User Reviews===
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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)

Latest revision as of 04:18, 26 September 2022

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K104001

The part was cloned into the Bacillus integration vector pJWV1012 upstream of both gfp and mCherry.

It was then integrated into the chromosome of Bacillus subtilis 168.

We showed that 168 was able to sense the levels of subtilin quorum communication peptide (SpaS product) in the external media.

The part can therefore be used as a generic sensor for subtilin mediated communication between Gram positive bacteria.

An ideal addition would be a resistance brick. Without the resistance genes, subtilin will kill Bacillus subtilis 168, causing cell lysis at high concentrations.

See the [http://2008.igem.org/Team:Newcastle_University Newcastle] 2007 wiki for more details .

User Reviews

UNIQ20d52e01e3a122c5-partinfo-00000000-QINU UNIQ20d52e01e3a122c5-partinfo-00000001-QINU


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)