Part:BBa_K2013017
2-oxopent-4-enoate hydratase
This part contains the sequence of encode 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase which catalyzes 2-hydroxymuconate semialdehyde into 2-oxopent-4-enoate.In addition，we did codon optimization before synthesizing it to encode the target product successfully in E. coli.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Experimental Validation
This part is validated through Four ways: amplification, PCR, Enzyme cutting and Sequence.
Amplification
Enzyme:KOD
Primer-F:5GAATTCGCGGCCGCTTCTAGAGTACTAGAGAAAGAGGAGAAATACTAGATGACCAACGTGAACG-3′
Primer-R:5′- CGCTACTAGTATTATTACCACGCGGTGTGATACAG-3′
Results
PCR
Enzyme:Taq
Primer-F:5′-CCACCTGACGTCTAAGAAAC-3′
Primer-R:5′-GTATTACCGCCTTTGAGTGA-3′
Results
Double digestion
After the assembly ,the plasmid was transferred into the Competent E. coli top10. After culturing overnight in LB,we minipreped the plasmid for double digestion .The first cutting procedure was performed with EcoRI and NcoI restriction endonuclease. The second cutting procedure was performed with PstI and NcoI restriction endonuclease.The plasmid was cutted in a 25μL system at 37 ℃ for 1 hours. The Electrophoresis was performed on a 1％ Agar
Results
| None |