Part:BBa_K4015007

CotA-Lcp-HlyA

CotA-Lcp-HlyA is composed of three basic parts, Lcp1VH2, HlyA and CotA. HlyA and Lcp are connected by Gibson assembly in order to allow Lcp to be secreted outside the cell through HlyA,and assembling CotA on the composite part can help to indicate the secretion.

Design consideration

As we introduce signal peptide into latex clearing protein production, we need a visual indicator to indicate the functioning and the presence of signal peptide secretion. GFP and laccase are the two reporters that have been used in our experiment. Vector p47 with GFP is used to examine the effectiveness of different peptides at first.

Later, we introduced lactase-cotA as our indicator. Laccase will only react with a specific substrate---ABTS. As we encode laccase into the cell, it will produce laccase along with Lcp. Since ABTS is filled into the LB, it will react with laccase that produced by the cell and soon be oxidized, turning into the color of bullish-green. Therefore, by using laccase, we will be allowed to observe the secretion of Lcp according to the color change.

The effect and result

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.

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. 

To examine the signal peptide’s capacity in secreting Lcp, we designed the plasmid pET23a-laccase-Lcp1VH2-HlyA and expressed in E.coli BL21.The expression of target protein after iptg induction showed a clear band in Fig.12. To test the activity of Lcp1VH2 secreted autonomously for rubber degradation, cis-1,4polyisoprene granules are mixed with culture fluid and the supernatant in a centrifuge tube respectively, and a change in dissolved oxygen content by time was measured by a dissolved oxygen probe.The culture fluid is the metabolites of E. coli BL21 pET23a::laccase-Lcp1VH2-hlya(including the secreted Lcp1VH2), the supernatant is the soluble protein after lysis and centrifugation. Results in Fig.12 shows that Lcp1VH2 in both supernatant and culture medium displayed oxygenase activity, and the concentration of Lcp1VH2 appears to be higher in the supernatant than in the culture medium.

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Fig 10. A.SDS-PAGE of crude extracts(C) and soluble fractions(S) of E. coli BL21(1) as control, Lcp1VH2 (2) and fusion proteins laccase-Lcp1VH2-hlya (3). B.Oxygen consumption reaction using supernatant and culture fluid of E.coli BL21 pET23a::laccase-Lcp1VH2-HlyA. 

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