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Latest revision as of 20:18, 19 October 2016
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
We also used BBa_J33204 to construct a device and demonstrated that it worked out well.
BBa_J33204 includes the xylE gene .This gene encodes the enzyme catechol-2,3-dioxygenase (metapyrocatechase), which also converts catechol to the bright yellow product 2-hydroxy-cis,cis-muconic semialdehyde.
