BBa_K2013017

BBa_K2013017 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_K2013017
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 08:16:58

2-oxopent-4-enoate hydratase



Types
DnaRegion

Roles
Translational_Unit

engineered_region

Sequences BBa_K2013017_sequence (Version 1)

Description

This part contains the sequence of coding 2-oxopent-4-enoate hydratase which catalyzes 2-hydroxymuconate semialdehyde into 2-oxopent-4-enoate.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:5GAATTCGCGGCCGCTTCTAGAGTACTAGAGAAAGAGGAGAAATACTAGATGACCAACGTGAACG-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 NcoI 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% Agar


Results

Notes

This is a Translational units part that encodes 2-oxopent-4-enoate hydratase

Source

This sequence is from Pseudomonas putida F1

Sequence Annotation Location Component / Role(s)
RBS
2-hydroxy-6-oxohepta-2,4-dienoate hydrolase
1,13
20,852
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_K2013017/1