Composite

Part:BBa_K4015009

Designed by: Andrew Peng Yi   Group: iGEM21_KEYSTONE   (2021-10-19)


CotA-HlyA

CotA-Hlya is composed of two basic parts, laccase, and HlyA. The composition enables the secretion with a clear indication within the culture plate with ABTS since the laccase can show bullish-green after its contact with ABTS.


Usage and Biology

We created p47-laccase-Lcp, p47-laccase-Lcp-HlyA, p47-laccase-HlyA, and pET28-Lcp-HlyA as the control to test our hypothesis. In the results, the control group (Figure 4A), as expected, showed no sign of coloration due to the absence of laccase; in the sample containing p47-laccase-Lcp (Figure 4B) secretion happened which implies that laccase by itself can act as a secretion agent; for p47-laccase-HlyA, no secretion happened, and we suspect that HlyA and laccase might suppress each other’s activity; however, when laccase and HlyA are positioned at the two ends of Lcp, this conflict is resolved, showed by Figure 4. As shown by figure 4, CotA-Lcp-HlyA has achieved successful secretion.


T--KEYSTONE--p23.png


Fig  4.    A. Strain containing p28a-lcp-hlyA; no coloration observed. B. Srtain containing p47-laccase-lcp; coloration was observed, indicating success in secretion. C. Strain containing p47-laccase-hlyA; no coloration observed. D. Strain containing p47-laccase-lcp-hlyA; coloration was observed, indicating success in secretion. 

citations:

1. Blaudeck, N., Sprenger, G. A., Freudl, R., & Wiegert, T. (2001). Specificity of signal peptide recognition in TAT-dependent bacterial protein translocation. Journal of Bacteriology, 183(2), 604–610. https://doi.org/10.1128/jb.183.2.604-610.2001

2. Freudl, R. (2018). Signal peptides for recombinant protein secretion in bacterial expression systems. Microbial Cell Factories, 17(1). https://doi.org/10.1186/s12934-018-0901-3

3.  Linton, E., Walsh, M. K., Sims, R. C., & Miller, C. D. (2011). Translocation of green fluorescent protein by comparative analysis with multiple signal peptides. Biotechnology Journal, 7(5), 667-676. https://doi.org/10.1002/biot.201100158 

4. Liu, J., Chen, J., Zuo, K., Li, H., Peng, F., Ran, Q., Wang, R., Jiang, Z., & Song, H. (2021). Chemically induced oxidative stress improved bacterial laccase-mediated degradation and detoxification of the synthetic dyes. Ecotoxicology and Environmental Safety, 226, 112823. https://doi.org/10.1016/j.ecoenv.2021.112823 

5. Janusz, G., Pawlik, A., Świderska-Burek, U., Polak, J., Sulej, J., Jarosz-Wilkołazka, A., & Paszczyński, A. (2020). Laccase properties, physiological functions, and evolution. International Journal of Molecular Sciences, 21(3), 966. https://doi.org/10.3390/ijms21030966

6. Yaohua, G., Ping, X., Feng, J., & Keren, S. (2019). Co-immobilization of laccase and ABTS onto novel dual-functionalized cellulose beads for highly improved biodegradation of indole. Journal of Hazardous Materials, 365, 118-124. https://doi.org/10.1016/j.jhazmat.2018.10.076


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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1348
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 286


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