Difference between revisions of "Part:BBa K4307045"

 
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<partinfo>BBa_K4307045 short</partinfo>
 
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Consisting of inducer nisin, membrane receptor NisK, regulatory protein NisR and promoter PnisA activated by nisR, nisin TCS is an important signal transduction system in lactobacillus. After the extracellular nisin combining with NisK, nisK will phosphorylate the regulatory protein NisR, which activates the promoter PnisA and starts the expression of downstream genes. <br>
  
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Here, we constructed the J23100-nisK-nisR+PnisA-EGFP composite part with pFB20 backbone and transplanted it into E. coli to achieve our goal of detecting proteins. This composite part consists two basic parts, J23100-nisK-nisR and PnisA-EGFP. J23100-nisK-nisR play the role of constitutive expressing the membrane receptor NisK and the regulatory protein NisR, of which J23100 is a strong constitutive promoter. PnisA-EGFP plays the role of receiving activation signals from nisR and expressing the reporter gene EGFP.
 
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===Usage and Biology===
 
===Usage and Biology===
  
 
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<span class='h3bb'>Sequence and Features</span>
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<h2>Sequence and Features</h2>
 
<partinfo>BBa_K4307045 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4307045 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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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-k4307045-gel.png" class="image">
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                <img alt="" src="https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307045-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-k4307045-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 J23100-nisK-nisR+PnisA-EGFP.</b>
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            </div>
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        </div>
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    </div>
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</div>
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<h3>Flow cytometry was done to characterize the biobrick.  </h3>
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<p>To measure the effectiveness of the nisin TCS, we added the gene of EGFP downstream of the promoter PnisA. If nisinTCS can function normally, the expression of downstream EGFP can be detected after addition of the inducer nisin.</p>
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<p>We conducted flow cytometry for 4h induction bacteria(Figure 2) to detect the expression of EGFP under induced and control conditions. Flow cytometry result shows positive percentage of experimental group induced by 1ng/ml Nisin is higher than control group, which means successful fluorescence induction.<br>
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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-k4307045.png" class="image">
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                <img alt="" src="https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307045.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-k4307045.png" class="internal" title="Enlarge"></a>
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                </div>
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                <b>Figure 2: </b> <b> pNisin2 nisin induction results by flow cytometry. </b>
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            </div>
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        </div>
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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>Through the above verification, we proved that our modified nisin TCS can function normally in <i>E.coli</i>. It can sense extracellular protein signals, amplify them and transform them into expression of downstream gene. By changing the proteins fused with nisin, Our modified nisin TCS can also be used to detect different proteins, which makes it have potential to be used by other iGEM teams.</p>
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</html>
  
  

Revision as of 15:50, 13 October 2022


NisK-NisR-PnisA-EGFP

Consisting of inducer nisin, membrane receptor NisK, regulatory protein NisR and promoter PnisA activated by nisR, nisin TCS is an important signal transduction system in lactobacillus. After the extracellular nisin combining with NisK, nisK will phosphorylate the regulatory protein NisR, which activates the promoter PnisA and starts the expression of downstream genes.

Here, we constructed the J23100-nisK-nisR+PnisA-EGFP composite part with pFB20 backbone and transplanted it into E. coli to achieve our goal of detecting proteins. This composite part consists two basic parts, J23100-nisK-nisR and PnisA-EGFP. J23100-nisK-nisR play the role of constitutive expressing the membrane receptor NisK and the regulatory protein NisR, of which J23100 is a strong constitutive promoter. PnisA-EGFP plays the role of receiving activation signals from nisR and expressing the reporter gene EGFP.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2934


Characterization

The following figure demonstrates our successful construction.


Figure 1: The construction results of J23100-nisK-nisR+PnisA-EGFP.

Flow cytometry was done to characterize the biobrick.

To measure the effectiveness of the nisin TCS, we added the gene of EGFP downstream of the promoter PnisA. If nisinTCS can function normally, the expression of downstream EGFP can be detected after addition of the inducer nisin.

We conducted flow cytometry for 4h induction bacteria(Figure 2) to detect the expression of EGFP under induced and control conditions. Flow cytometry result shows positive percentage of experimental group induced by 1ng/ml Nisin is higher than control group, which means successful fluorescence induction.

Figure 2: pNisin2 nisin induction results by flow cytometry.

Conclusion

Through the above verification, we proved that our modified nisin TCS can function normally in E.coli. It can sense extracellular protein signals, amplify them and transform them into expression of downstream gene. By changing the proteins fused with nisin, Our modified nisin TCS can also be used to detect different proteins, which makes it have potential to be used by other iGEM teams.