Help:Standards/Assembly/RFC23
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Reason: This assembly standard is not accepted for the iGEM competition
Motivation & Discussion
The Silver RFC[23] is a modification of the BioBrick RFC[10] that allows for in-frame protein assembly. It relies on shortening the BioBrick prefix and suffix each by removing their spacer base. When parts are assembled by cutting XbaI and SpeI and ligating their overhangs together, a 6bp scar is formed. Since the restriction sites of the prefix and suffix are the same, the assembly protocols and restriction enzymes for BioBrick RFC[10] are used for Silver RFC[23].
The Silver RFC[23] addresses some of the concerns of the BioBrick RFC[10]: the scar is 6bp allowing for in-frame assembly and does not include a stop codon. However, RFC[23]'s 6bp scar includes an Arginine codon, which is destabilizing (N-end rule) resulting in faster degradation. Additionally the spacer bases, which are removed in Silver RFC[23], are meant to prevent Dam methylation. As such parts beginning in "TC" create a site capable of inhibiting XbaI cutting, so precautions must be made to allow for these parts to follow the RFC[23] prefix.
Advantages
- in-frame assembly of protein parts
- restriction-compatible to the BioBrick RFC[10] -- no new enzymes
Disadvantages
- N-terminal Thr-Arg = destabilization signal (N-end rule)
- Dam methylation blocks cloning when prefix is followed by "TC"
- unexpected side-effects for users not aware of the shortened prefix/suffix
- non-coding parts may be not functionally compatible due to the changed bp distance
- frameshift with respect to what is expected from protein coding original BioBrick assembly standard parts
- not possible to preserve native protein start (but equivalent to original BioBrick assembly standard coding part could be constructed)
Technical Specifications
Prefix and Suffix
Prefix Suffix
5' - GAATTC GCGGCCGC T TCTAGA ...part... ACTAGT A GCGGCCG CTGCAG - 3'
EcoRI NotI XbaI SpeI NotI PstI
Scar
Assembling two parts leaves the following scar:
5' [part A] ACTAGA [part B] 3'
T R
Compatability/Illegal Sites
In order for a part to be compatible with Silver RFC[23] 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
- SpeI site: ACTAGT
- PstI site: CTGCAG
- NotI site: GCGGCCGC
Notes
Sources
- [http://dspace.mit.edu/handle/1721.1/32535 A New Biobrick Assembly Strategy Designed for Facile Protein Engineering]
- [http://openwetware.org/wiki/The_BioBricks_Foundation:Standards/Technical/Formats#BBF_RFC_25_.28aka_Fusion_parts.2C_Freiburg_iGem2007_team.29 The BioBricks Foundation - Format Discussion]
- Help:Assembly_standard_23
Accepted Standards: BioBrick RFC[10] | iGEM Type IIS RFC[1000]