Help:Assembly standard 23

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SilverFusionScheme.png

Pam Silver's lab has developed Assembly standard 23, often called the Silver standard, for assembling protein domains. It relies on shortening the BioBrick prefix and suffix each by 1 base pair such that the resulting SpeI-XbaI scar is only 6 base pairs long and protein domains can be assembled in frame.

As shown in figure 3 (right), Assembly standard 23 results in the inclusion of a Thr-Arg (ACT AGA) two amino acid junction between protein domains.

One disadvantage of the Assembly standard 23 is that the codon AGA which encodes Arginine is a rare codon in E. coli. Rare codons can prevent over-expression of proteins in E. coli.

See The BioBricks Foundation wiki for a discussion and comparison of different technical standards.

Advantages

  • in-frame fusion of protein parts
  • restriction-compatible to the original BioBrick assembly standard parts -- no new enzymes
  • also protein parts can, theoretically, be fused N-terminally to original BioBrick assembly standard protein parts, as long as the frameshift is corrected by an adapter part
  • used by several iGEM teams

Disadvantages

  • Arg in scar can be problematic
  • 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)

References

A New BioBrick Assembly Strategy Designed for Facile Protein Engineering
MIT SBWG Technical Reports, 2006 Apr 20
Ira Philips, Pam Silver
URL (open access!)