Team:NAU-CHINA/Basic Part



Best Basic Part

BBa_K2088000

This basic part encodes Catechol 2,3-Dioxygenase from Sphingobium sp. YBL2. This protein transforms catechol by meta-cleavage to the bright yellow product 2-hydroxymuconate semialdehyde(2-HMS). We tested it with J23100 (a strong promoter) and its native rbs before it and a double terminator after it in E.coli. It plays a great role in our project to degrade catechol to 2-HMS, a non-toxic molecule that can be metalbolized by E.coli DH5α.



Verification of the function of catechol 2,3-dioxygenase from YBL2

Background

Catechol is a toxic organic metabolite found in the degradation of pesticide. Catechol 2,3-dioxygenase can rapidly convert catechol into 2-hydroxymuconic semialdehyde (2-HMS). 2-HMS is a non-toxic, bright yellow molecule that can be metalbolized by E.coli DH5α.

Fig.1

Bacterial Strain

Strain Sphingomonads sp.YBL2 was cultured on LB medium with streptomycin(100mg/L) at 30℃ for 2 days.

Cloning of catechol 2,3-dioxygenase(C23O) gene with native RBS

Primer: Forward primer: GAATTCGCGGCCGCTTCTAGAGGCTGCCTGAACAAGACTGAG

Reverse primer: CTGCAGCGGCCGCTACTAGTAACGCCACAGGTTTAGAAGC

Fig.2

PCR System(15µL):

template single colonies
ddH2O 6.9ul
Primer 0.5ul
MixEx TaqTM Version 7.5ul
2.0 plus dye

Total time: 1h49min

Fig.3

PCR System(50µL):

template single colonies/bacteria liquid (2µL)
ddH2O 23µL or 21µL
Primer 2µL
MixPrime STAR 25µL

Total time: 48min

Fig.4

Construction of Recombinant Expression Vector

Fig.5

Functional Verification

Method

Step one: Transformed the vector into E.coli (DH5α) and streaked it on LB agar with chloramphenicol.

Step two: Dripped 100μL 0.2mol/L catechol onto the colonies, and placed at 37℃ for 10 minutes.

*Note that the catechol solution should be kept away from light and air.

The picture below shows the plate before and after dripping catechol solution. It indicated that 2-HMS was produced by C23O.

*pbaC as negative control (CK).

Fig.6

Fig.7