Difference between revisions of "Help:Standards/Assembly/RFC25"

Revision as of 20:25, 28 February 2012

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?

Related Links

• Standards
• 3A Assembly
• Gibson Assembly

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]
• Help:Assembly_standard_25