Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Binglin Liu
Date created: 2016-10-11 11:00:00
Date modified: 2016-10-12 11:26:25
RBS and MHETase
| Types | DnaRegion |
| Roles | engineered_region Translational_Unit |
| Sequences | BBa_K2013003_sequence (Version 1) |
Description
This part contains the coding sequence of MHETase which can hydrolyzes MHET to TPA and EG. MHETase is second enzymes on the downstream of PETase in the bacterium Ideonella sakaiensis 201-F6 that Japanese scientists found.MHET is degraded into two kinds of natural environment harmless substances: terephthalic acid and ethylene glycol. We carried out a codon optimization on this part.In addition，We carried out codon optimization on this part.Experimental Validation
This part is validated through Four ways: amplification, PCR, Enzyme cutting and Sequence.
Amplification
Enzyme:Q5
Primer-F:5′- GAATTCGCGGCCGCTTCTAGAGTACTAGAGTCACACAAAAGGGTACTAGATG-3′
Primer-R:5′- CGCTACTAGTATTATTACGGCGGAGCCGCGCAC-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 HindIII restriction endonuclease. The second cutting procedure was performed with PstI and BaHI 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