Difference between revisions of "Part:BBa K4307010"

 
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Originated from <i>Lactococcus lactis subsp. Lactis</i>, NisA is the precursor of nisin A, a lanthionine-containing peptide antibiotic (lantibiotic) active on Gram-positive bacteria. Affibody Z<sub>EGFR:2377</sub> is derived from derived from staphylococcal protein A through directly evolution.<sup>[1]</sup> We managed to design and express NisA-affibody fusion protein.<br>
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Functioned together with NisB(BBa_K4307011) and NisC(BBa_K4307012), it forms composite part NisA-affi-NisB+NisC (BBa_K4307046) to produce functional NisA-affibody peptide.
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<span class='h3bb'>Sequence and Features</span>
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<h2>Sequence and Features</h2>
 
<partinfo>BBa_K4307010 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4307010 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-k4307010-gel.png" class="image">
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                <img alt="" src="https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307010-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-k43070010-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 NisA-affi.</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>Western blots was done to characterize the bio brick.  </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-k4307010.png" class="image">
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                <img alt="" src="https://static.igem.wiki/teams/4307/wiki/wiki-parts-files/mainpage/bba-k4307010.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-k4307010.png" class="internal" title="Enlarge"></a>
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                </div>
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                <b>Figure 2: </b> <b> Western blot results to detect NisA-affibody expression.</b>
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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>According to the western blots result, it can be found that NisA-affi can be successfully expressed in <i>E.coli</i>, which indicates that precursor nisin fusion protein can be produced and purified. This ensures the effectiveness of functional NisA-affibody production in <i>E.coli</i>.</p>
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<h2>References</h2>
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[1] Friedman, M., Orlova, A., Johansson, E., Eriksson, T. L., Höidén-Guthenberg, I., Tolmachev, V., Nilsson, F. Y., & Ståhl, S. (2008). Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule. <i>Journal of molecular biology</i>, 376(5), 1388–1402.
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Latest revision as of 16:31, 13 October 2022


nisA-affi

Originated from Lactococcus lactis subsp. Lactis, NisA is the precursor of nisin A, a lanthionine-containing peptide antibiotic (lantibiotic) active on Gram-positive bacteria. Affibody ZEGFR:2377 is derived from derived from staphylococcal protein A through directly evolution.[1] We managed to design and express NisA-affibody fusion protein.
Functioned together with NisB(BBa_K4307011) and NisC(BBa_K4307012), it forms composite part NisA-affi-NisB+NisC (BBa_K4307046) to produce functional NisA-affibody peptide.


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 NisA-affi.

Western blots was done to characterize the bio brick.

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: Western blot results to detect NisA-affibody expression.

Conclusion

According to the western blots result, it can be found that NisA-affi can be successfully expressed in E.coli, which indicates that precursor nisin fusion protein can be produced and purified. This ensures the effectiveness of functional NisA-affibody production in E.coli.

References

[1] Friedman, M., Orlova, A., Johansson, E., Eriksson, T. L., Höidén-Guthenberg, I., Tolmachev, V., Nilsson, F. Y., & Ståhl, S. (2008). Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule. Journal of molecular biology, 376(5), 1388–1402.