Difference between revisions of "Part:BBa K4307008"

 
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Originated from <i>Lactococcus lactis subsp. Lactis</i>, NisR is a member of the two-component regulatory system NisK/NisR involved in the regulation of the biosynthesis of lantibiotic nisin. NisR functions as a regulatory protein which is phosphorylated by NisK in response to environmental nisin signals. We managed to express NisR in <i>E.coli</i> strain.<br>
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Functioned together with NisK(BBa_K4307007), PnisA(BBa_K4307009), J23100 and EGFP, it forms composite part J23100-nisK-nisR+PnisA-EGFP(BBa_K4307045) to express fluorescence signal induced by nisin.
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<span class='h3bb'>Sequence and Features</span>
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<h2>Sequence and Features</h2>
 
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<!-- Uncomment this to enable Functional Parameter display  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K4307008 parameters</partinfo>
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<partinfo>BBa_K4307000 parameters</partinfo>
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<h2>Characterization</h2>
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<p>The following figure demonstrates our successful construction.</p>
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<br>
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<div class="center">
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    <div class="thumb tnone">
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        <div class="thumbinner" style="width:50%;">
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            <a href="https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307008-gel.png" class="image">
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                <img alt="" src="https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307008-gel.png" width="100%" height=auto class="thumbimage" /></a>                  <div class="thumbcaption">
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                <div class="magnify">
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                    <a href="https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307008-gel.png" class="internal" title="Enlarge"></a>
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                </div>
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                <b>Figure 1: </b> <b>The construction results of NisR.</b>
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            </div>
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        </div>
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    </div>
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<h3>SDS-PAGE assay was done to characterize the biobrick.</h3>
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<p>To determine the expression of NisR, we used T7 promoter and lac operator to control the overexpression of NisR by IPTG. NisR in bacterial lysate was detected by SDS-PAGE(Figure 2), indicating that NisR can be successfully expressed in <i>E.coli</i>.  </p>
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<p>
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  <br>
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<div class="center">
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    <div class="thumb tnone">
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        <div class="thumbinner" style="width:50%;">
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            <a href="https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307008.png" class="image">
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                <img alt="" src="https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307008.png" width="100%" height=auto class="thumbimage" /></a>                  <div class="thumbcaption">
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                <div class="magnify">
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                    <a href="https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307008.png" class="internal" title="Enlarge"></a>
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                </div>
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                <b>Figure 2: </b> <b> SDS-PAGE result of NisR expression.</b>
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            </div>
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</div>
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</p>
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<h2>Conclusion</h2>
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<p>According to the SDS-PAGE result, it can be found that NisR can be successfully expressed in <i>E.coli</i>, which indicates that it can act as the downstream regulator of NisK to pass down nisin signal functionally in <i>E.coli</i> as in <i>Lactococcus lactis</i>. This ensures the effectiveness of nisin TCS in <i>E.coli</i>.</p>
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</html>
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===Functional Parameters===
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<partinfo>BBa_K4307000 parameters</partinfo>
 
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Latest revision as of 16:28, 13 October 2022

NisR

Originated from Lactococcus lactis subsp. Lactis, NisR is a member of the two-component regulatory system NisK/NisR involved in the regulation of the biosynthesis of lantibiotic nisin. NisR functions as a regulatory protein which is phosphorylated by NisK in response to environmental nisin signals. We managed to express NisR in E.coli strain.
Functioned together with NisK(BBa_K4307007), PnisA(BBa_K4307009), J23100 and EGFP, it forms composite part J23100-nisK-nisR+PnisA-EGFP(BBa_K4307045) to express fluorescence signal induced by nisin.


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]


Characterization

The following figure demonstrates our successful construction.


Figure 1: The construction results of NisR.

SDS-PAGE assay was done to characterize the biobrick.

To determine the expression of NisR, we used T7 promoter and lac operator to control the overexpression of NisR by IPTG. NisR in bacterial lysate was detected by SDS-PAGE(Figure 2), indicating that NisR can be successfully expressed in E.coli.


Figure 2: SDS-PAGE result of NisR expression.

Conclusion

According to the SDS-PAGE result, it can be found that NisR can be successfully expressed in E.coli, which indicates that it can act as the downstream regulator of NisK to pass down nisin signal functionally in E.coli as in Lactococcus lactis. This ensures the effectiveness of nisin TCS in E.coli.