Results

RESULTS

In silico Design

The laccase gene was codon optimized for expression in E.coli.
The laccase gene and pET26b expression vector were analyzed and appropriate restriction endonucleases were selected.

Recombinant DNA was modeled by simulating digestion and ligation processes with restriction enzyme on Benchling.
Each of these processes was performed separately for wild-type laccase, pelB.laccase and dsb.laccase

Plasmid Amplification by Transformation

The pUC cloning plasmids carrying the laccase gene (wild-type, dsb.laccase and pelB.laccase) were transformed into E. coli DH5α cells by heat shock method and colony selection performed on ampicillin agar plates.

Plasmid Isolation

Plasmids cloned into E.coli DH5α cells were isolated with the ABM Column-Pure Plasmid Miniprep Kit. The VisualLyse indicator was used to fully understand that bacterial lysis occurred.
The isolation results were measured with nanodrop (Thermofisher).

Cutting with Restriction Endonucleases

The pUC plasmids containing the laccase gene and the pET26b expression vector were with HindIII and NdeI enzymes..

The digested pET26b and laccase genes isolated from the gel. Then, isolated fragments were measured on the nanodrop and checked on the gel.

Since another band anormally was imaged on the gel and it was difficult to distinguish with the help of ladder due to the proximity of the bands, both bands continued to work until sequencing results were obtained. These fragments are called heavy and light.

Sample ID	Nucleic Acid Unit	A260(Abs)	A280(Abs)	260/280	260/230
dpET26b 1	50.4 ng/µl	1.008	0.719	1.4	0.27
dpET26b 2	0.8 ng/µl	1.016	-0.041	-0.39	0
dsb light	26.1 ng/µl	0,521	0.242	2.15	0.08
dsb heavy	48 ng/µl	0.94	0.547	1.72	0.15
wild-type light	47.4 ng/µl	0.947	0.585	1.62	0.12
wild-type heavy	32.9 ng/µl	0.65	0.297	2.22	0.06
pelB light	8.9 ng/µl	0.179	0.048	3.75	0.01
pelB heavy	8.9 ng/µl	0.177	0.047	3.77	0.02

Ligation

The DNA fragments isolated from the gel after digestion were ligated with T4 DNA Ligase enzyme.

Recombinant DNA cloned into E.coli DH5 cells for amplifiying, was isolated and transformed into E.coli SHuffle cells for expression.

After transformation, transformant colonies were selected on LBA medium containing antibiotics.

Protein Isolation

E.coli SHuffle cells including the recombinant laccase gene were induced by IPTG overnight for expression of laccase enzymes

After expression, polyhistidine tagged laccase enzymes were isolated with magnetic tube rack (GeneOn) and the MagneHis Protein Purification System (Promega).