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

Latest revision as of 17:20, 6 September 2019

Motivation & Discussion

The Freiburg 2007 iGEM Team designed the Freiburg RFC[25] as an extension of the BioBrick RFC[10] that allows for in-frame protein assembly, while also avoiding some concerns with the Silver RFC[23].

RFC[25] adds a start codon and a restriction site (NgoMIV) to the prefix and a restriction site (AgeI) followed by a stop codon in the suffix, while maintaining the restriction sites of the BioBrick [RFC] prefix and suffix. When NgoMIV and AgeI are cut their overhangs are compatible forming a Thr-Gly scar: a protein domain, missing a start and stop codon (as these have have been incorporated into the prefix and suffix), can be assembled to another protein domain.

Since RFC[25] uses a start codon within its prefix followed by NgoMIV this will affect the N-terminus of the protein coding region. Some proteins may have issues with such a modification to their N-terminus. In cases where the native N-terminus needs to be preserved, a "N-part" format, which uses the BioBrick RFC[10] CDS prefix, is used instead.

Note: Freiburg RFC[25] does not adhere to the Registry's paradigm of protein domains and protein assembly.

The Freiburg RFC[25] is compatible with both 3A Assembly and Standard Assembly (with some changes in protocol). Scarless assembly, like Gibson, will work as well, and is encouraged as it can make for scarless protein assembly, and avoid RFC[25]'s exception to N-parts.

Find RFC25 parts in the RFC25 BioBricks collection.

See the [http://dspace.mit.edu/handle/1721.1/45140 Freiburg proposal] for more information.

Advantages

• in-frame assembly 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 Silver RFC[23] protein parts (frame shift + stop codon), without special considerations

Technical Specifications

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 

Scar

Assembling two parts leaves the following scar:

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

N-Parts Prefix and Suffix

For "N-parts"

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

Compatability/Illegal Sites

In order for a part to be compatible with the Freiburg RFC[25] 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

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]
• Help:Assembly_standard_25