Team:KEYSTONE/Contribution


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Overview

This year we expressed a new latex clearing protein (Lcp1 VH2) and enhanced its yield and solubility by NusA and made it autocrine by laccase and HlyA to achieve a more convenient and sustainable process of Lcp degradation of rubber.

But we encountered the problem of protein expression at the beginning. We tried the method in the literature, but it didn't work.We optimized the expression system of Lcp1 VH2 protein for subsequent igemer.

In addition,we added new data to iGEM's existing part—laccase (Part:BBa_K2684000).


A effective expression system for Lcp1 VH2

According to the papers we referenced, Lcp1 VH2 is usually inserted into the pET23a plasmid and subsequently transferred into the E.coli C41 strain for expression.

We tried that approach, but the bands of Lcp1 VH2 were indistinguishable and could not tell whether it was expressed in E.coli C41(Fig.1).In order to obtain a viable expression system, we tested four expression protocols using existing strains on hand (E.coli C41 and E.coli BL21) with vectors (pet23a and pet28a): E.coli C41 pET23a:: Lcp1 VH2, E.coli C41 pET28a:: Lcp1 VH2, E.coli BL21 pET23a:: Lcp1 VH2, E.coli BL21 pET28a:: Lcp1 VH2 and E.coli BL21 pET28a:: Lcp1 VH2 E.coli BL21 pET23a:: Lcp1 VH2 and E.coli BL21 pET28a:: Lcp1 VH2. as shown in Figure 2,we can see that E.coli BL21 pET23a:: Lcp1 VH2 is the best system for Lcp1 VH2 expression: compared to the control, we can see a clear induction band and the Lcp1 VH2 expression and water solubility were excellent.

The author also claimed the applying of AIM(Auto-Induction Medium) would determine the induction time completely on glucose concentration (which can avoid biomass monitoring and culture manipulation) in the cultivation media and the inhibition caused by the glucose can be compared to the optimal addition time of inducers. However, The medium of AIM is relatively hard to obtain with complex composition and laborious preparation process.

Eventually we found Lcp1 VH2 is expressed effectively when inserted into pET23a in traditional expression system(E.coli BL21) with IPTG induction in LB——the most common medium.

Fig 1. Optimization of the expression system of Lcp1 VH2. SDS-PAGE of crude extracts(C) and soluble fractions(S) . A. Expression of

VH2 and pET28a::Lcp1 VH2 in E.coli C41: E.coli C41(1) as control, pET23a:: Lcp1 VH2 (2)and pET28a:: Lcp1 VH2 (3). B. pET23a:: Lcp1 VH2 and expression of pET28a:: Lcp1 VH2 in E.coli BL21: E.coli BL21(4) as control, pET28a:: Lcp1 VH2 (5) and pET23a:: Lcp1 VH2 (6).


New characterization of Laccase

Add new data to iGEM's existing part—laccase (Part:BBa_K2684000). We found that laccase has an autocrine function and can also act as a fusion chaperone to assist in the secretion of the target protein. This was not mentioned in the old part page. And laccase is a good indicator when it acts as a fusion partner, and it can react with ABTS when it is secreted into solid medium. 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 (Fig 2A), as expected, showed no sign of coloration due to the absence of laccase; in the sample containing p47-laccase-Lcp (Fig 2B) 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(Fig 2C); however, when laccase and HlyA are positioned at the two ends of Lcp, this conflict is resolved(Fig 2D).

Fig 2. 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.




References

Altenhoff, A. L., Thierbach, S., & Steinbüchel, A. (2020). High yield production of the latex clearing protein from Gordonia polyisoprenivorans VH2 in fed batch fermentations using a recombinant strain of Escherichia coli. Journal of biotechnology, 309, 92–99. https://doi.org/10.1016/j.jbiotec.2019.12.013
Andler, R., Heger, F., Andreeßen, C., & Steinbüchel, A. (2019). Enhancing the synthesis of latex clearing protein by different cultivation strategies. Journal of biotechnology, 297, 32–40. https://doi.org/10.1016/j.jbiotec.2019.03.019

Ilcu, L., Röther, W., Birke, J., Brausemann, A., Einsle, O., & Jendrossek, D. (2017). Structural and Functional Analysis of Latex Clearing Protein (Lcp) Provides Insight into the Enzymatic Cleavage of Rubber. Scientific reports, 7(1), 6179. https://doi.org/10.1038/s41598-017-05268-2

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

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