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> | 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> | ||
| − | + | Functioned together with NisB(BBa_K4307011) and NisC(BBa_K4307012), it forms composite part NisA-affi-NisB+NisC (BBa_K4307014) to produce functional NisA-affibody peptide. | |
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<partinfo>BBa_K4307010 SequenceAndFeatures</partinfo> | <partinfo>BBa_K4307010 SequenceAndFeatures</partinfo> | ||
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<partinfo>BBa_K4307000 parameters</partinfo> | <partinfo>BBa_K4307000 parameters</partinfo> | ||
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<h2>Characterization</h2> | <h2>Characterization</h2> | ||
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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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<b>Figure 1: </b> <b>The construction results of NisA-affi.</b> | <b>Figure 1: </b> <b>The construction results of NisA-affi.</b> | ||
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<h3>Western blots was done to characterize the bio brick. </h3> | <h3>Western blots was done to characterize the bio brick. </h3> | ||
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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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<b>Figure 2: </b> <b> Western blot results to detect NisA-affibody expression.</b> | <b>Figure 2: </b> <b> Western blot results to detect NisA-affibody expression.</b> | ||
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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. | [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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<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
Revision as of 11:22, 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_K4307014) to produce functional NisA-affibody peptide.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization
The following figure demonstrates our successful construction.
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.
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.