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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)

Emerson reaction assay (4-aminoantipyrine colorimetric reaction)

Emerson reaction is describing 4-aminoantipyrine (4-AAP) oxidation with phenol in alkaline condition catalyzed by oxidative potassium ferricyanide (K3[Fe(CN)6]) to produce p-quinoneimide adduct in red color.

Emerson reaction is describing 4-aminoantipyrine (4-AAP) oxidation with phenol in alkaline condition catalyzed by oxidative potassium ferricyanide K3[Fe(CN)6] to produce p-quinoneimide adduct in red color.

To test Emerson reaction, we prepare phenol solution (83g/L, equals to 0.88M, i.e., phenol solubility in water) with 10X serial dilution. The red color product of the reaction can be measured at OD580. The color changed from yellow, orange to red color depends on the concentration of phenol.

The calibration curve presented in the figure below is made in the scatter plot. The concentration of phenol (mg/L) (Y) can be calculated from the values of OD580 by the equation of Y = 0.00003*e^(16.202*X)

CYP2E1 functional assay

Benzene is one of the substrates of CYP2E1. Because of the toxicity of benzene, a collegiate iGEM team, Tunghai-TAPG helped us conduct the analysis in a specialized laboratory in the Department of Chemistry in the university.

Experiment procedure

↓ prepare CYP2E1 and WT lysates
↓ prepare 1.79 g/L of benzene (i.e., benzene solubility in water)
↓ add 90μl of benzene solution with 10X serial dilution to each well
↓ incubate with 40μl of CYP2E1 or WT Bacillus lysates at room temperature for 30 min
↓ transfer 90μl of the mixture to a new well
↓ add 90μl of solution I (1% of 4-aminoantipyrine in KOH solution, pH=9~10
↓ then add 45μl of solution II (4% of K3[Fe(CN)6])
↓ measure at OD580

The data showed that the values measured at OD580 are higher in the group of benzene plus CYP2E1 compared to controls of benzene without CYP2E1, indicating that the phenol is converted from benzene by CYP2E1. The benzene has a basal effect in Emerson reaction. The values of OD580 of Benzene are regarded as background and are subtracted for study the phenol generation.

Next, according to the calibration curve of phenol analysis in Emerson reaction. We converted the OD values of phenol formation to benzene degradation. The result suggested CYP2E1 in the bacterial lysate can convert 0.09 - 0.47 μg/L of benzene to phenol, implying the possibility of CYP2E1 application in benzene removal in our microalgae purification system.