Difference between revisions of "Help:Standards/Assembly/RFC25"

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==Philosophy==
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The Freiburg 2007 iGEM Team designed the Freiburg Standard RFC[25] as an extension of the BioBrick Standard RFC[10], which adds two restriction sites while maintaining the BioBrick sites in the prefix and suffix. The motivation for the Freiburg Standard is to allow for in-frame protein fusions, while also avoiding some of the issues with the Silver Standard.
  
 +
 +
===Advantages===
 +
* in-frame fusion of protein parts
 +
* benign protein scar
 +
* N-end rule safe (long protein half-life)
 +
* provision for preserving native N-terminal while using RBS parts
 +
* both new enzymes can be heat-inactivated
 +
* stand-alone protein expression (start + stop in prefix / suffix)
 +
* full BBa compatibility -- functionally & compositionally equivalent to BBa protein coding part
 +
* blunt-cutting isochizomer of NgoMIV (NaeI) -- possibility of directional cloning with two inner restriction sites enables part transfer between different formats and other potentially interesting transfer reactions.
 +
 +
===Disadvantages===
 +
* N-parts are assembled with a different enzyme combination.
 +
* not compatible to BioFusion protein parts (frame shift + stop codon), but see below!
 +
 +
 +
==Technical Specifications==
 +
===Compatability/Illegal Sites===
 +
In order for a part to be compatible with the Freiburg Standard 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
 +
*NgoMIV site: GCCGGC
 +
*AgeI site: ACCGGT
 +
*SpeI site:    ACTAGT
 +
*PstI site:    CTGCAG
 +
*NotI site:    GCGGCCGC
 +
 +
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===Prefix and Suffix===
  
 
               Prefix                                    Suffix
 
               Prefix                                    Suffix
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Assembling two parts leaves the following scar:
 
Assembling two parts leaves the following scar:
  
−
  5' [part A] [part B] 3'
+
  5' [part A] ACTAGA [part B] 3'
 +
 
 +
 
 +
===Primers===
 +
 
 +
 
 +
==Notes?==
 +
===Related Links===
 +
*Standards
 +
*3A Assembly
 +
*Gibson Assembly
 +
 
 +
 
 +
===Sources===
 +
*[http://dspace.mit.edu/handle/1721.1/45140 Fusion Protein (Freiburg) Biobrick assembly standard]
 +
*[http://openwetware.org/wiki/The_BioBricks_Foundation:Standards/Technical/Formats#BBF_RFC_25_.28aka_Fusion_parts.2C_Freiburg_iGem2007_team.29 The BioBricks Foundation:Standards/Technical/Formats BioBrick BB-2]
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*[[Help:Assembly_standard_25]]
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Revision as of 20:24, 28 February 2012

Philosophy

The Freiburg 2007 iGEM Team designed the Freiburg Standard RFC[25] as an extension of the BioBrick Standard RFC[10], which adds two restriction sites while maintaining the BioBrick sites in the prefix and suffix. The motivation for the Freiburg Standard is to allow for in-frame protein fusions, while also avoiding some of the issues with the Silver Standard.


Advantages

  • in-frame fusion of protein parts
  • benign protein scar
  • N-end rule safe (long protein half-life)
  • provision for preserving native N-terminal while using RBS parts
  • both new enzymes can be heat-inactivated
  • stand-alone protein expression (start + stop in prefix / suffix)
  • full BBa compatibility -- functionally & compositionally equivalent to BBa protein coding part
  • blunt-cutting isochizomer of NgoMIV (NaeI) -- possibility of directional cloning with two inner restriction sites enables part transfer between different formats and other potentially interesting transfer reactions.

Disadvantages

  • N-parts are assembled with a different enzyme combination.
  • not compatible to BioFusion protein parts (frame shift + stop codon), but see below!


Technical Specifications

Compatability/Illegal Sites

In order for a part to be compatible with the Freiburg Standard 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
  • NgoMIV site: GCCGGC
  • AgeI site: ACCGGT
  • SpeI site: ACTAGT
  • PstI site: CTGCAG
  • NotI site: GCGGCCGC


Prefix and Suffix

             Prefix                                     Suffix
5' - GAATTC GCGGCCGC T TCTAGA TG GCCGGC...part... ACCGGT TAAT ACTAGT A GCGGCCG CTGCAG - 3'
     EcoRI    NotI      XbaI     NgoMIV            AgeI        SpeI     NotI    PstI

Assembling two parts leaves the following scar: 

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


Primers

Notes?

Related Links

  • Standards
  • 3A Assembly
  • Gibson Assembly


Sources

  • [http://dspace.mit.edu/handle/1721.1/45140 Fusion Protein (Freiburg) Biobrick assembly standard]
  • [http://openwetware.org/wiki/The_BioBricks_Foundation:Standards/Technical/Formats#BBF_RFC_25_.28aka_Fusion_parts.2C_Freiburg_iGem2007_team.29 The BioBricks Foundation:Standards/Technical/Formats BioBrick BB-2]
  • Help:Assembly_standard_25


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