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Part name:CYP2E1, rabbit cytochrome P450 2E1 ( optimized for Bacillus preferred codon)

Part cat. no.:BBa_K2932000

Biological function

Benzene oxidation

Phenol is a product from benzene oxidation which is catalyzed by CYP2E1.

Gene cloning and protein expression

We optimized the gene codon of rabbit cytochrome P450 2E1 (CYP2E1) based on Bacillus subtilis codon usage preference and synthesized the DNA fragment by Twist Bioscience. Then, DNA fragments were amplified by PCR and assembled with a terminator, followed by inserting to PliaI-RBS/pSB1C3.

To express CYP2E1 gene in Bacillus subtilis, we transfer the DNA fragments of PliaI-RBS-CYP2E1-Tr to pBS0E vector and transformed Bacillus subtilis 168 with the resulting plasmids.

Protein induction procedure

↓ culture Bacillus subtilis 168 carrying the plasmid of PliaI + RBS + CYP2E1 + terminator/pBS0E in LB + Amp (100 μg/ml) O/N at 37°C, shaking at 170 rpm, supplemented with 1mM of thiamine and 75mg/L of 5-aminolevulinic acid
↓ transfer 3 ml to 50 ml LB+Antibiotics with all the supplements in 250 ml flask
↓ measure OD650
↓ shake at 200rpm,37°C until OD650 between 0.5~0.7
↓ add 50 μl of 30ug/ml Bacitracin for induction at 25°C, shaking at 100 rpm for 18.5 hr

After protein induction by bacitracin, the total lysates of Bacillus expressing CYP2E1 were subjected to SDS-PAGE and stained with Coomassie blue. CYP2E1 protein has a molecular weight of 55kDa and shown as a band overlapped with a lot of proteins located between 48-63 kDa. Further confirmation is needed to make sure the expression of CYP2E1 and optimal induction procedure. (The work of SDS-PAGE was collaborated with team NCTU-Formosa)