Difference between revisions of "Part:BBa K302018"
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<partinfo>BBa_K302018 short</partinfo>
 
<partinfo>BBa_K302018 short</partinfo>
  
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Subtilin is a lantibiotic peptide produced naturally by ''Bacillus subtilis'' ATCC 6633 but not by ''Bacillus subtilis'' 168. This part encodes two separate subtilin immunity systems, which together provide a high degree of subtilin resistance.  
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Subtilin is a lantibiotic peptide produced naturally by ''Bacillus subtilis'' ATCC 6633 but not by ''Bacillus subtilis'' 168.
  
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This part encodes two separate subtilin immunity systems &mdash; a membrane bound protein that binds subtilin and prevents it from penetrating the cell membrane to form pores, and a transporter which removes subtilin from the membrane &mdash; which together provide a high degree of subtilin resistance.
  
 
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<partinfo>BBa_K302018 parameters</partinfo>
 
<partinfo>BBa_K302018 parameters</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). The result was documented in the experience page and the main page of  (<partinfo>BBa_K302018</partinfo>).<br>
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===Contribution from iGEM2022 ZJU-China===
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<p>The sequence of  (<partinfo>BBa_K302018</partinfo>) was synthesized and cloned into the PHY-plasmid to obtain the recombinant expression vector </p>   
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<p>We first tested the inhibitory concentration of subtilisin by <b>zone of inhibition experiment</b>, using BL21 transformed by PHY-Immunity plasmid or PHY-Empty plasmid as control. The concentration of subtilisin were 100mg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml. </p>
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<div align="center">[[File:GLL-2.png|600px]]</div>
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<center>Fig1 Zone of inhibition experiment for subtilisin in BL21.
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</center>
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<p>Then, we prepared normal LB culture plates with different concentration of subtilisin, including 0.1mg/ml, 0.2mg/ml, 1mg/ml and 10mg/ml, in individual dishes. Then we inoculated BL21 transformed by immunity plasmid or PHY-empty plasmid as control in the plates. For the immunity group, some colonies grew on the surface of the media with 1mg/ml subtilisin, while the control group didn’t. This suggested that the PHY-Immunity plasmid worked well in BL21.</p>
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<div align="center">[[File:GLL-1.png|600px]]</div>
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<center>Fig2 The colonies growth condition in plates with different concentration of subtilisin

Latest revision as of 03:51, 26 September 2022

Subtilin immunity

Subtilin is a lantibiotic peptide produced naturally by Bacillus subtilis ATCC 6633 but not by Bacillus subtilis 168.

This part encodes two separate subtilin immunity systems — a membrane bound protein that binds subtilin and prevents it from penetrating the cell membrane to form pores, and a transporter which removes subtilin from the membrane — which together provide a high degree of subtilin resistance.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 837
    Illegal BglII site found at 1585
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1852
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1132
    Illegal SapI site found at 1412
    Illegal SapI.rc site found at 2135


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). The result was documented in the experience page and the main page of (BBa_K302018).

Contribution from iGEM2022 ZJU-China

The sequence of (BBa_K302018) was synthesized and cloned into the PHY-plasmid to obtain the recombinant expression vector

We first tested the inhibitory concentration of subtilisin by zone of inhibition experiment, using BL21 transformed by PHY-Immunity plasmid or PHY-Empty plasmid as control. The concentration of subtilisin were 100mg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml.

GLL-2.png
Fig1 Zone of inhibition experiment for subtilisin in BL21.

Then, we prepared normal LB culture plates with different concentration of subtilisin, including 0.1mg/ml, 0.2mg/ml, 1mg/ml and 10mg/ml, in individual dishes. Then we inoculated BL21 transformed by immunity plasmid or PHY-empty plasmid as control in the plates. For the immunity group, some colonies grew on the surface of the media with 1mg/ml subtilisin, while the control group didn’t. This suggested that the PHY-Immunity plasmid worked well in BL21.

GLL-1.png
Fig2 The colonies growth condition in plates with different concentration of subtilisin