Part:BBa_K4998018

Laccase gene (silA)

Usage and Biology

Laccases are enzymes that belong to the superfamily of Multicopper Oxidases (MCOs). Their most commonly known characteristic is their particularly wide substrate specificity since it can include polyphenolic compounds, lignin and lignin-like structures, humus, etc. This is the reason why they can be used in biotechnology, like in bioremediation. The type of substrate oxidized depends on the pH of the environment. About their structure, they usually consist of three domains, but two-domain laccases have been identified as well. Laccases contain four copper ions that are distributed to three copper centers. That gives them the ability to use molecular oxygen as their final electron acceptor and have the ability to reduce oxygen to water but without the production of harmful byproducts.

Laccases can be produced by fungi, plants, bacteria mostly those in soil or aquatic environments, animals, and even some archaea. Fungal laccases have been widely studied and used since their discovery, but recently the scientific community has taken an interest in bacterial laccases due to their properties. [1]

In our project, Euphoresis, we take advantage of the bacterial laccase lac7 [2] which is expressed by the silA gene originating the bacterial strain from Streptomyces ipomonae, that is characterized as a small and thermostable protein. Our goal is to oxidase phenolic and non-phenolic aromatic compounds in the soil after wildfires.

Sequence and Features

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 326
  Illegal XhoI site found at 199
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 130
  Illegal NgoMIV site found at 792
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 76
  

Characterization

PCR and Agarose Gel Electrophoresis

After the first assembly attempt in the pJUMP29-1D'(sfGFP) backbone, following the 3A assembly protocol in E. coli competent cells, the silA gene fragment was amplified using PCR, under the primers: BBa_K4998002, BBa_K4998003. After PCR amplification, Agarose gel electrophoresis was conducted, and the laccase's column exhibited a band in the 200bp regions, confirming the successful silA gene assembly into the backbone.

Figure 1. Agarose gel electrophoresis (3A Assembly)

A second assembly attempt in the same plasmid backbone, but under the Modified Assembly Protocol with the use of two restriction enzymes, PstI and EcoRI, for E. coli competent cells, the above PCR procedure was conducted and the outcome was examined with Agarose Gel Electrophoresis:

Figure 2. Agarose gel electrophoresis (Modified Assembly Protocol Assembly)

Once more, a band was shown in the 200bp region, implying successful silA gene assembly.

Bradford assay

The Bradford assay was conducted to determine the amount of total protein under different CuSO4 concentrations. After calibration, the following standard curve is made:

Figure 3.

Figure 4

After implementing the Bradford Assay, the following results are obtained:

Figure 5

Figure 6

As it is proved, the raise of CuSO₄ concentrations cause a protein production rise. Since CuSO₄ is related to the copper functioning laccase enzyme, it can be implied that a rise of laccase production is detected when this molecule is present in higher concentrations.

SDS-PAGE

The silA gene's laccase is a ~75 kDa protein [1, 2]. An SDS-PAGE was conducted to determine the presence of the laccase enzyme in the transformed Bacillus subtilis WB800N strain. The following results were obtained:

Figure 7. SDS-PAGE for the lac7 enzyme in the transformed B. subtilis WB800N strain, under different CuSO₄ concentrations. The bands at the ~75 kDa imply lac7 presence in the extracellular B. subtilis region.

The produced laccase in the extracellular region was quantificated via band absorbtion measurement, under the same CuSO₄ concentrations as in the Bradford Assay. In this way,t he total protein concentration at ~75 kDa band region was measured. The following diagram has been obtained:

Figure 8. SDS-PAGE lac7 quantification diagram

A notable protein concentration raise, in the range of 75 kDa, was detected, implying a boosted laccase production as CuSO₄ concentration rises.

References

[1] 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

[2] Yang, L.-H., Qiao, B., Xu, Q.-M., Liu, S., Yuan, Y., & Cheng, J.-S. (2021). Biodegradation of sulfonamide antibiotics through the heterologous expression of laccases from bacteria and investigation of their potential degradation pathways. Journal of Hazardous Materials, 416, 125815. https://doi.org/10.1016/j.jhazmat.2021.125815