Help:Standards/Assembly/RFC23

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


More About Assembly Standards

Help:Assembly Standards || Assembly Compatibility || Supported Assembly Systems
BioBrick RFC[10] | BioBrick BB-2 RFC[12] | Berkeley RFC[21] | Silver RFC[23] | Freiburg RFC[25]