Difference between revisions of "Part:BBa K4307007"

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Originated from <i>Lactococcus lactis subsp. Lactis</i>, NisK is a member of the two-component regulatory system NisK/NisR involved in the regulation of the biosynthesis of lantibiotic nisin. NisK functions as a membrane-associated protein kinase that phosphorylates NisR in response to environmental nisin signals. We managed to express NisK in <i>E.coli</i> strain.<br>
 
Originated from <i>Lactococcus lactis subsp. Lactis</i>, NisK is a member of the two-component regulatory system NisK/NisR involved in the regulation of the biosynthesis of lantibiotic nisin. NisK functions as a membrane-associated protein kinase that phosphorylates NisR in response to environmental nisin signals. We managed to express NisK in <i>E.coli</i> strain.<br>
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Functions together with NisR(BBa_K4307008), PnisA(BBa_K4307009), J23100 and EGFP, it forms composite part J23100-nisK-nisR+PnisA-EGFP(BBa_K4307013) to express fluorescence signal induced by nisin.
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Functioned together with NisR(BBa_K4307008), 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>
 
<partinfo>BBa_K4307007 SequenceAndFeatures</partinfo>
 
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<p>The following figure demonstrates our successful construction.</p>
 
<p>The following figure demonstrates our successful construction.</p>
  
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                 <a href="https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307007-gel.png" class="image">
 
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                     <img alt="" src="https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307007-gel.png" width="100%" height=auto class="thumbimage" /></a>                  <div class="thumbcaption">
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                     <b>Figure 1: </b> <b>The construction results of NisK.</b>   
 
                     <b>Figure 1: </b> <b>The construction results of NisK.</b>   
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The PCR screening was checked with 0.6% agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the
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NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel). Colonies 1, 2 and 3
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have shown the right size.
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<h3>Fluorescence location assay was done to characterize the biobrick.</h3>
 
<h3>Fluorescence location assay was done to characterize the biobrick.</h3>
 
<p>To determine whether the constitutively expressed NisK in our system could locate on cell membrane correctly, we design NisK-EGFP fusion protein, expressed the protein in MACH1-T1 strain and examined its cellular location under confocal laser scanning microscope. The imaging result showed stronger fluorescence signal located on bacterial membrane, and fluorescence intensity measured by computer also showed the same distribution(Figure 2), indicating that most NisK-EGFP fusion proteins were located correctly on the membrane.  </p>
 
<p>To determine whether the constitutively expressed NisK in our system could locate on cell membrane correctly, we design NisK-EGFP fusion protein, expressed the protein in MACH1-T1 strain and examined its cellular location under confocal laser scanning microscope. The imaging result showed stronger fluorescence signal located on bacterial membrane, and fluorescence intensity measured by computer also showed the same distribution(Figure 2), indicating that most NisK-EGFP fusion proteins were located correctly on the membrane.  </p>
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https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307007.png<br>
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                <b>Figure 2: </b> <b> NisK localization under confocal laser scanning microscope.</b>
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                    <b>Figure 2: </b> <b> NisK localization under confocal laser scanning microscope.</b>
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<h2>Conclusion</h2>
 
<h2>Conclusion</h2>
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<p>According to the location assay data, it can be found that NisK can locate on membrane correctly in <i>E.coli</i>, which indicates that it can act as a membrane receptor to sense 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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<p>According to the location assay data, it can be found that NisK can locate on membrane correctly in <i>E.coli</i>, which indicates that it can act as a membrane receptor to sense 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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Latest revision as of 16:27, 13 October 2022


NisK

Originated from Lactococcus lactis subsp. Lactis, NisK is a member of the two-component regulatory system NisK/NisR involved in the regulation of the biosynthesis of lantibiotic nisin. NisK functions as a membrane-associated protein kinase that phosphorylates NisR in response to environmental nisin signals. We managed to express NisK in E.coli strain.
Functioned together with NisR(BBa_K4307008), 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 NisK.

Fluorescence location assay was done to characterize the biobrick.

To determine whether the constitutively expressed NisK in our system could locate on cell membrane correctly, we design NisK-EGFP fusion protein, expressed the protein in MACH1-T1 strain and examined its cellular location under confocal laser scanning microscope. The imaging result showed stronger fluorescence signal located on bacterial membrane, and fluorescence intensity measured by computer also showed the same distribution(Figure 2), indicating that most NisK-EGFP fusion proteins were located correctly on the membrane.


Figure 2: NisK localization under confocal laser scanning microscope.

Conclusion

According to the location assay data, it can be found that NisK can locate on membrane correctly in E.coli, which indicates that it can act as a membrane receptor to sense nisin signal functionally in E.coli as in Lactococcus lactis. This ensures the effectiveness of nisin TCS in E.coli.