Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Laura van Smeden
Date created: 2015-09-02 11:00:00
Date modified: 2015-09-18 05:38:08
Outer Membrane Protein X (OmpX) with BamHI-linker and mNeonGreen
| Types | DnaRegion |
| Roles | CDS Coding |
| Sequences | BBa_K1761001_sequence (Version 1) |
Description
This part consists of three subparts, namely the uter membrane protein OmpX, a BamHI linker and the fluorophore mNeonGreen.OmpX is an outer membrane protein with the C- and N-termini in the intracellulair domain. To be able to use OmpX as a scaffold, a non-natural amino acid needs to be introduced. This can be done by implementing the amber stop codon TAG in one of the loops of OmpX via a mutation. With a specific tRNA an azide-functionalized amino acid can be built in, which can be used for the SPAAC click chemistry reaction with DBCO functionalized groups.
The BamHI linker is a 213 bp long flexible GGSGGS linker. Using the restriction enzyme BamHI, the linker can become 45 bp shorter.
mNeonGreen is a yellow-green fluorescent protein. For a fluorescene measurement, the mNeonGreen fluorophore can be excitated with a laser with a wavelenght of 480 nm and readed out at 517 nm.
Notes
The sequence is redesigned considering codon optimalization. Also restriction sites for biobricking and classical cloning are deleted. From the BamHI linker, the coding sequecne for GGSGGS is redesigned causing that only two BamHI restriction sites remain.Source
OmpX is a outer membrane protein naturally occuring in wildtype E.coli.The BamHI linker is inspired by the article "Quantitative Understanding of the Energy Transfer between Fluorescent Proteins Connected via Flexible Peptide Liners" by Toon H. Evers et all from 29 August 2006.
mNeonGreen naturally occurs in the organism Branchiostoma Lanceolatum.
| Sequence Annotation | Location | Component / Role(s) |
| OmpX BBa_K1761000 mNeonGreen BBa_K1761003 BamHI-linker | 1,513 1,513 727,1437 727,1437 514,726 | feature/protein CDS engineered_region feature/BioBrick feature/protein CDS engineered_region feature/BioBrick feature/dna sequence_feature |