BBa_K2013003

BBa_K2013003 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_K2013003
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

Notes

This is a Translational units part encoding MHETase.

Source

This sequence is from a bacterium called Ideonella sakaiensis 201-F6

Sequence Annotation Location Component / Role(s)
RBS
MHETase
1,13
20,1842
feature/rbs ribosome_entry_site
CDS feature/protein
igem#experience
None
 
igem#status
Planning
 
synbiohub#ownedBy
user/james
 
synbiohub#ownedBy
user/myers
 
synbiohub#topLevel
BBa_K2013003/1