Part:BBa_K2455003

Cell-Penetrating USER Cassette

This biobrick contains an AsiSI/Nb.BSMI USER Cassette with a N-terminal nona-arginine (R9) Cell-penetrating peptide which gives proteins inserted into the cassette the ability to pass through plasma membranes. In addition the USER Cassette carries a N-terminal hexa-histidine tag which allows for easy purification.

For our study we demonstrated that proteins inserted into the cassette can be purified, using the histidine-tag, and subsequently transferred into E. coli cells through the use of the R9 peptide.

In addition; previous studies have shown proteins associated with R9, either covalently or non-covalently, to be able to enter a variety of cell types (Chang et al.).

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

Cell-Penetrating Peptides (CPPs) are small peptides, typically rich in basic residues, which are able to facilitate transport of a wide variety of cargoes across plasma membranes. Their origin in nature comes from viral domains such as the viral HIV tat domain (Eudes & Chugh, 2008). In recent years research have been done into making synthetic CPPs, especially peptides constructed solely from arginine residues have been of interest. The arginine rich sequence has been shown to trigger endocytosis in a wide range of cell types, including onion and potato cells (Chang et al.).

Gene Insertion

In order to test the ability of the biobrick to facilitate protein import in E. coli we choose to insert two different fluorescent proteins into it, SYFP2 and mTag BFP.

Opening of USER Cassette

To facilitate insertion we first had to prepare the vector, this was done in a two step digestion reaction. First to open the cassette 42 µL purified vector was mixed with 2.5 µL AsiSI, 10 µL 10x Tango Buffer, and 45.5 µL Nuclease-free water. The mixture was then incubated for 3 hours at 37 degrees Celsius. 5 µL linearised vector was run for 20 min at 100 V on a 1 % Agarose gel to check for proper linearisation.

Billede af CPP digestion

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Evt. oven over column purification og Nb.BSMI digestion