BBa_K2013009

BBa_K2013009 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_K2013009
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 08:12:35

TPA 1,2-dioxygenase



Types
DnaRegion

Roles
Translational_Unit

engineered_region

Sequences BBa_K2013009_sequence (Version 1)

Description

This part contains one of sequences coding TPA 1,2-dioxygenase.This is the second. TPA is incorporated through the TPA transporter (TPATP) and catabolized by TPA 1,2-dioxygenase (TPADO).The result is that TPA is catabolized into 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate.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:KOD


Primer-F:5′-GAATTCGCGGCCGCTTCTAGAGTACTAGAGTCACACAGGAGAGTACTAGATG-3′


Primer-R:5′- CGCTACTAGTATTATTACAGCGGCAGCGCCA-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 NcoI restriction endonuclease. The second cutting procedure was performed with PstI and EcoRV 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 relates to encode TPA 1,2-dioxygenase

Source

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

Sequence Annotation Location Component / Role(s)
RBS
ISF6_0228
1,13
20,487
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_K2013009/1