Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Demin Xu
Date created: 2016-10-11 11:00:00
Date modified: 2016-10-15 08:15:41
catechol 2，
3dioxygenase
| Types | DnaRegion |
| Roles | Translational_Unit engineered_region |
| Sequences | BBa_K2013016_sequence (Version 1) |
Description
This part contains the sequence of coding catechol 2，3dioxygenase which can catalyze catechol into 2-hydroxymuconate semialdehyde.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:5′-GAATTCGCGGCCGCTTCTAGAGTACTAGAGTCACACAGGAGGGTACTAGATG-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 PstI restriction endonuclease. The second cutting procedure was performed with KpnI 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 catechol 2，3dioxygenaseSource
This sequence is from Escherichia coli Strain MG1655| Sequence Annotation | Location | Component / Role(s) |
| RBS dioxygenase | 1,13 20,1171 | feature/rbs ribosome_entry_site CDS feature/protein |