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Freiburg Standard RFC[25]

Philosophy

The Freiburg 2007 iGEM Team designed the Freiburg Standard RFC[25] as an extension of the BioBrick Standard RFC[10], which adds two restriction sites while maintaining the BioBrick sites in the prefix and suffix. The motivation for the Freiburg Standard is to allow for in-frame protein fusions, while also avoiding some of the issues with the Silver Standard.

Advantages

in-frame fusion of protein parts
benign protein scar
N-end rule safe (long protein half-life)
provision for preserving native N-terminal while using RBS parts
both new enzymes can be heat-inactivated
stand-alone protein expression (start + stop in prefix / suffix)
full BBa compatibility -- functionally & compositionally equivalent to BBa protein coding part
blunt-cutting isochizomer of NgoMIV (NaeI) -- possibility of directional cloning with two inner restriction sites enables part transfer between different formats and other potentially interesting transfer reactions.

Disadvantages

N-parts are assembled with a different enzyme combination.
not compatible to BioFusion protein parts (frame shift + stop codon), but see below!

Technical Specifications

Compatability/Illegal Sites

In order for a part to be compatible with the Freiburg Standard it must not contain the following restriction sites, as these will need to be unique to the prefix and suffix:

EcoRI site: GAATTC
XbaI site: TCTAGA
NgoMIV site: GCCGGC
AgeI site: ACCGGT
SpeI site: ACTAGT
PstI site: CTGCAG
NotI site: GCGGCCGC

Prefix and Suffix

             Prefix                                     Suffix
5' - GAATTC GCGGCCGC T TCTAGA TG GCCGGC...part... ACCGGT TAAT ACTAGT A GCGGCCG CTGCAG - 3'
     EcoRI    NotI      XbaI     NgoMIV            AgeI        SpeI     NotI    PstI

Assembling two parts leaves the following scar:

5' [part A] ACTAGA [part B] 3'

Primers

Notes?

Sources

[http://dspace.mit.edu/handle/1721.1/45140 Fusion Protein (Freiburg) Biobrick assembly standard]
[http://openwetware.org/wiki/The_BioBricks_Foundation:Standards/Technical/Formats#BBF_RFC_25_.28aka_Fusion_parts.2C_Freiburg_iGem2007_team.29 The BioBricks Foundation:Standards/Technical/Formats BioBrick BB-2]
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