Part:BBa_K2455002

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 SYFP2 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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

The R9-SYFP2-H6 biobrick was constructed by amplifying SYFP2 using primers with overhangs designed for insertion into the USER cassette. The PCR product was then purified and ligated into the vector, before being restriction digested and sequenced.

Biobrick Creation

In order to create the improved version of SYFP2 we inserted it into the Cell-Penetrating USER Cassette biobrick.