Help:Standards/Assembly/RFC21
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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
- [http://dspace.mit.edu/handle/1721.1/46747 BBF RFC 21: BglBricks Assembly Standard]
- [http://openwetware.org/wiki/The_BioBricks_Foundation:Standards/Technical/Formats#BBF_RFC_21:_The_Berkeley_.28BBb.29_Format_.28now_called_BglBricks.29 The BioBricks Foundation - Format Discussion]
- Old Registry RFC[21] Page
Accepted Standards: BioBrick RFC[10] | iGEM Type IIS RFC[1000]