Difference between revisions of "Part:BBa K2455002"

Revision as of 00:10, 2 November 2017

CPP-SYFP2

This is an improved version of the Super Yellow Fluorescent Protein 2 (BBa_K864100) carrying an N-terminal nona-arginin tag, and a C-terminal hexa-histidine tag. The R9-tag is a Cell-Penetrating Peptide giving its associated protein the ability to pass through plasma membranes.

For our study we demonstrated that this improved version of BBa_K864100 had fluorescence of similar levels to the original protein, and was able to stain both Escherichia coli and Pseudomonas putida cells, presumably through internalisation of the protein.

Previous studies have shown R9 associated proteins to be able to enter a variety of cell types (Chang et al., 2005).

Sequence and Features

Usage and Biology

SYFP2

SYFP2 is a monomeric GFP-based protein giving a bright yellow fluorescence. It has an excitation peak at 515 nm, an emission peak at 527 nm and a relative brightness compared to EYFP2(Q96K) of 12.0 in E. coli.

This version of SYFP2 has an N-terminal R9-tag and a C-terminal H6-tag.

Cell-Penetrating Peptides

Cell-Penetrating Peptides (CPPs) are small peptides, that are able to facilitate transport of a wide variety of cargoes across plasma membranes. CPPs are typically rich in basic residues and originate from viral domains such as the viral HIV tat domain (Eudes & Chugh, 2008). In recent years researchers have tried to make synthetic CPPs, with special interest in peptides constructed solely from arginine residues. The arginine rich sequences have been shown to trigger endocytosis in a wide range of cell types, including onion and potato cells (Chang et al., 2005).

Biobrick Creation

In order to create this improved biobrick SYFP2 inserted into the Cell-Penetrating USER Cassette biobrick.

Gene Amplification