Help:Standards/Assembly/RFC10

Motivation & Discussion

The BioBrick RFC[10], developed by Tom Knight, is a standard for interchangeable parts based on idempotent assembly. BioBrick RFC[10] is currently the most commonly used assembly standard: the majority of parts in the Registry database are RFC[10] compatible, and the majority of samples in the Registry's Repositories are maintained in RFC[10] plasmid backbones. As such, using RFC[10] ensures a greater diversity when designing your synthetic biology projects.

BioBrick RFC[10] uses an alternate shortened prefix for protein coding regions to prevent a rbs-cds issues.

A major concern with BioBrick RFC[10] is that it does not facilitate the assembly of proteins, the traditional 8bp scar creates a frame-shift, while the alternate 6bp scar includes a stop codon. If you wish to assemble proteins you will want to use an RFC designed specifically for protein assembly (see below). Alternatively, scarless assembly or DNA synthesis can circumvent this issue in RFC[10].

Advantages

  • standard
  • well tested and documented
  • native protein start codon can be preserved while using RBS parts.
  • large and still growing set of parts

Disadvantages

  • no protein fusion (stop codon in 6bp scar, frameshift and stop codon in 8bp scar)

Technical Specifications

Prefix and Suffix

             Prefix                                     Suffix
5' - GAATTC GCGGCCGC T TCTAGA G ...part... T ACTAGT A GCGGCCG CTGCAG - 3'
     EcoRI    NotI      XbaI                  SpeI     NotI   PstI 

There is a second prefix designed for protein coding regions (parts start with ATG, see RBS-CDS issues for more info):

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


Scar

Assembling two parts leaves the following scar:

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


The scar between a RBS and protein coding region (alternate prefix) would be:

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


Compatibility/Illegal Sites

In order for a part to be compatible with BioBrick RFC[10] it must not contain the following restriction sites, as these are unique to the prefix and suffix:

Sequence Type Enzyme Note
gaattc Illegal EcoRI
tctaga Illegal XbaI
actagt Illegal SpeI
ctgcag Illegal PstI
gcggccgc Avoid NotI


Notes

Sources

TomKnightPhoto.jpg
Tom Knight, a senior research scientist at MIT CSAIL, developed Assembly standard 10 in 2003. It is the most widely adopted assembly standard in synthetic biology.

References

  1. Idempotent Vector Design for Standard Assembly of Biobricks by Tom Knight
  2. BBF RFC 9: Idempotent vector design for the standard assembly of Biobricks by Tom Knight, Randall Rettberg, Leon Chan, Drew Endy, Reshma Shetty, Austin Che
  3. BBF RFC10: Draft standard for Biobrick biological parts by Tom Knight

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]