Difference between revisions of "Part:BBa K3589105"

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Mutant laccase from Botrytis aclada for Escherichia coli

This basic part contains the coding sequence of the mutant form L499F of the laccase from the ascomycete Botrytis aclada (hereafter referred to as BaLac). This part is codon-optimized for Escherichia coli. Combined a promoter and a terminator, this basic part mediates the oxidation of a wide variety of substrates including phenolic compounds and aromatic amines. This part can be cloned into standard E coli. expression vectors like the commercially available pGEX-6P-1 vector. The mutant laccase L499F has a high redox potential and shows activity near neutral pH (Scheiblbrandner et. al, 2017; doi: 10.1038/s41598-017-13734-0).

Summary of the Results from Team Kaiserslautern 2019 for part BBa_K3589105

• Expression could be achieved in different E. coli strains when cloned in the pGEX-6P-1 expression vector
• Protein showed activity in ABTS-assay
• Protein could degrade Diclofenac

Design of the constructs

For the recombinant expression of the laccase genes from Botritis aclada(baLac) the E. coli vector pGEX-6P-1 was used (Fig. 1). This expression vector is used to construct a translation fusion protein of Glutathione S-transferase (GST) and our two different laccases. The expression is regulated by a tac promotor. This promotor combines the strong expression rate from the tryptophan promotor and can be induced with IPTG like the lac operon. To make sure the promotor is inhibited if there is no induction with IPTG the vector also includes the genetic code for the lac-inhibitor that can bind the lac-operon. Because GST has a high affinity for glutathione, the fusion of the laccases with GST allows the purification by affinity chromatography using glutathione agarose. In addition, a protease cleavage site is incorporated between the GST and our fusion protein (BaLac-GST). This allows the separation of GST and the laccase using PreScission Protease. BaLac has a size of 89.3 kDa with GST and a size of 61.6 kDa without GST. For selection of plasmid containing cells the expression vector carries an ampicillin resistance gene.
In order to be able to do further enzyme assays with the laccases, the vector pGEX-6P-1-balac has to be transformed in the E. coli expression strain BL21(DE3). Additionally, we transformed them in E. coli DH5α for isolation the vectors. The strain has an endA1 mutation. This leads to the inactivity of an intracellular endonuclease, which degrades plasmid DNA. Therefore, plasmid DNA isolation is more efficient.

Fig. 1: pGEX-6P-1_baLac . It includes a tac promotor that can be regulated with the lac-inhibitor (expressed with lacI) which can be inactivated with IPTG. The protein is fused to GST, with a PreScission protease cleavage site in between. The vector has an ampicillin resistance gene, which is used for selection.

Growth test
First, we wanted to find out the best conditions for the plasmid containing E. coli BL21(DE3) cells to produce BaLac and marLac. We did a test expression where the cells grow at different temperatures (37°C, 30 °C and 17°C; Fig. 2). After every hour we took a sample for an SDS-PAGE (Fig. 3) and measured the optical density (OD) at 600 nm. We designed the experiment based on Kittl et al., 2012 where they used copper sulfate in the media for the protein production. So, we tested the growth at all the temperatures, one time with CuSO₄ and one time without, to be sure that the copper sulfate doesn’t influence the growth (Fig. 2).
To see if our protein is soluble or insoluble, we lysed the cells and separated the pellet and the soluble fraction with SDS-PAGE (Fig.3).

Fig. 2: Growth curve from E. coli BL21(DE3) pGEX-6P-1_baLac producing cells at different temperatures. The expression was done over 19 hours, the cells were induced at x=0. The growth of E. coli BL21(DE3) pGEX-6P-1_baLac is shown. It was tested at 37°C, 30°C and 17°C and all temperatures both with [w] and without CuSO₄.

Fig. 3: SDS-PAGE of the test expression with different temperatures. Samples of E.coli BL21(DE3) pGEX-6P-1_baLac were taken before induction and after inducing with IPTG after every hour for every temperature. The LB medium contains CuSO₄.The cells were disrupted by sonication and insoluble and soluble fraction were separated. The red boxes show the produced and BaLac. Marker: New England BioLabs ® Blue Protein Standard Broad Range. The proteins sought are at the level of a relative molecular mass of the marker between 75 and 100 kDa (BaLac, size 89.3 kDa) and 75 kDa (marLac, size 75.8 kDa). The positive control is a GST fusion protein with a size of 26 kDa.

Furthermore, we wanted to test in which medium the cells grow best. We compared LB-Medium with 2YT-Medium in two different temperatures (37°C, 30°C; Fig. 4)

Fig. 4: SDS-PAGE and western blot of the test expression with LB medium and 2YT medium. (A) shows BaLac producing cells growing in LB medium, (B) shows BaLac producing cells growing in 2YT medium. Each medium contains CuSO₄. After induction, the cells grow at 37 °C and 30 °C for 3 h and 5 h. The cells were disrupted by sonification and insoluble and soluble fraction were separated. The red boxes show the produced BaLac. The fusion protein (BaLac and GST) was detected by anti-GST-antibodies (first antibody) and anti-Goat alkaline phosphatase conjugated antibodies (second antibody). Marker: New England BioLabs ® Blue Protein Standard Broad Range.