Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Binglin Liu
Date created: 2016-10-11 11:00:00
Date modified: 2016-10-13 12:22:17
DCDDH
| Types | DnaRegion |
| Roles | Translational_Unit engineered_region |
| Sequences | BBa_K2013010_sequence (Version 1) |
Description
This part codes 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase(DCDDH).DCDDH is a important enzyme for our project,which can hydrolyze 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate to protocatechuate. 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′-GAATTCGCGGCCGCTTCTAGAGTACTAGAGTCACACAAAAGGGTACTAGATG-3′
Primer-R:5′- CGCTACTAGTATTATTACGCGGTGCCTTCCTCAAC-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