Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Mei Deng
Date created: 2016-10-11 11:00:00
Date modified: 2016-10-15 08:17:36
2-oxopent-4-enoate hydratase
| Types | DnaRegion |
| Roles | Translational_Unit engineered_region |
| Sequences | BBa_K2013018_sequence (Version 1) |
Description
This part contains the sequence of coding 2-oxopent-4-enoate hydratase that catalyzes 2-oxopent-4-enoate into 4-hydroxy-2-oxopentenoate.In addition，We carried out codon optimization on this partExperimental Validation
This part is validated through Four ways: amplification, PCR, Enzyme cutting and Sequence.
Amplification
Enzyme:KOD
Primer-F:5GAATTCGCGGCCGCTTCTAGAGTACTAGAGAAAGAGGAGAAATACTAGATGACCAAGCACACCC-3′
Primer-R:5′- CGCTACTAGTATTATTAGCTCAGGCTGCCTTTCGGCG-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 EcoRV 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％ Agarose glu.
Results
Notes
This is a Translational units part that encodes 2-oxopent-4-enoate hydrataseSource
This sequence is from Escherichia coli P12b| Sequence Annotation | Location | Component / Role(s) |
| RBS 2-oxopent-4-enoate hydratase | 1,13 20,831 | ribosome_entry_site feature/rbs CDS feature/protein |