Help:Standards/Assembly/RFC21

Motivation & Discussion

The Berkeley RFC[21] is optimized to enable in-frame assembly of proteins: it is based on idempotent assembly with BamHI and BglII restriction enzymes, which creates a benign 6bp scar between assembled parts.

The Berkeley RFC[21] is incompatible with BioBrick RFC[10]. Additionally the BglII enzyme cannot be heat inactivated, which prevents the RFC[21] from being used with the 3A assembly method.

Advantages

  • in-frame assembly of protein parts
  • benign protein scar
  • enzymes selected for efficient cutting

Disadvantages

  • BglII cannot be heat-inactivated therefore the current 3A Assembly procedures won't work
  • incompatible to BioBrick RFC[10] format
  • incompatible to Silver RFC[23] format


Technical Specifications

Prefix and Suffix

        Prefix                        Suffix
5' GAATTC atg AGATCT ...part... GGATCC taa CTCGAG 3'
   EcoRI      BglII             BamHI   *   XhoI 


Scar

Assembling two parts leaves the following scar:

5' [part A] GGATCT [part B] 3'
             G  S


Compatibility/Illegal Sites

In order for a part to be compatible with Berkeley RFC[21] it must not contain the following restriction sites, as these will need to be unique to the prefix and suffix:

  • EcoRI site: GAATTC
  • BglII site: AGATCT
  • BamHI site: GGATCC
  • XhoI site: CTCGAG


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]