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The following is adapted from the [http://parts.mit.edu/igem07/index.php/Freiburg07/report_fusion_parts 2007 Albert-Ludwigs Universitat Freiburg iGEM team wiki].

RFC

• [http://hdl.handle.net/1721.1/45140 BBF RFC 25: Fusion Protein (Freiburg) Biobrick assembly standard] by Kristian M. Müller, Katja M. Arndt, the 2007 Freiburg iGEM team, and Raik Grünberg

Background

The generalized BioBrick prefix and suffix with its easy cloning strategy is an excellent and universal way to combine various parts, e.g. promoter region, gene of interest, terminator etc. However, it is not well-suited for in-frame assembly of protein domains due to the 8 bp SpeI/XbaI scar.

Suffix of the first part (part in gray, PstI, NotI, SpeI):

...ACtactagtagcggccgctgcag

combined with Prefix of the second part (EcoRI, NotI, XbaI,part in gray):

gaattccgcggccgcttctagagCA...

results after an SpeI/Xba combination in:

...ACtactagagCA...

encoding the amino acids Tyr (codon: tac), STOP (codon: tag) and Ser or Arg (codon agn).

Consequently, protein domains cannot be designed as separate BioBrick parts and assembled together. The Freiburg assembly standard was developed to address this shortcoming while retaining compatibility with the original BioBrick version. The Freiburg standard extends the original BioBrick standard with two additional compatible restriction sites to allow for the modular assembly of protein fusion parts. For the Freiburg standard, appropriate enzymes were chosen carefully to include ensure coding for amino acids which are compatible with flexible linkers as well as with the N-end rule for protein stability.

Specification

The Freiburg assembly standard extends the original BioBrick suffix and prefix with two additional compatible restriction sites. The frame of the standard suffix and prefix remains the same resulting in complete compatibility with any previously designed parts. We chose the restriction sites NgoMVI and AgeI as they code for the amino acids Ala-Gly or Thr-Gly, respectively, which are compatible with flexible linkers commonly used in fusion proteins and also compatible with the N-end rule for protein stabiliy. Consequently, we name these parts FusionParts. Assembly of two parts that adhere to the Freiburg assembly standard creates an AgeI/NgoMIV scar coding for Thr-Gly, which can easily be integrated in any linker sequence. Furthermore, the NgoMIV site (coding for Ala-Gly) after the start Methionine of the standard BioBrick suffix adheres completely to the N-end rule. For proteins, which are sensitive to amino acid addition at the N-terminus, we also devised an N-Part with a suffix that lacks the NgoMIV site.

The following list summarizes the most important aspects of the Freiburg assembly standard.

• Both parts, the FusionPart and the N-part are fully compatible with all standard iGEM parts as they have the BioBricks prefix for coding sequences and the standard BioBrick suffix.
• Both parts have two additional enzymes, NgoMIV and AgeI, which have compatible cohesive ends and enable in-frame fusion of protein parts with the linker sequence TG (no stop codons).
• The only difference of the N-part and FusionPart is the additional NgoMIV site in the FusionPart.
• The FusionPart is the universal part for fusion proteins, and it can be a stand-alone protein part as it has a start codon after the XbaI site (BioBrick prefix for coding sequence), with two additional amino acids (A, G) encoded before the start of the protein.
• The N-part is designed to be the start of a fusion protein or a stand-alone protein part, in which the N-terminus is sensitive to any amino acid addition, to be cloned via XbaI/PstI to any iGEM RBS expression part.
• The FusionPart can be fused to the N-part by digesting the N-part with AgeI/SpeI and the FusionPart with NgoMIV/SpeI.
• Any number of FusionParts can be combined and optionally fused to the N-part.
• nnnnnn is a place holder for the coding sequence of the respective part.

Prefix

Freiburg standard FusionPrefix (EcoRI, NotI, XbaI, NgoMIV, part in gray; original BioBrick prefix for coding sequences underlined):

gaattccgcggccgcttctagatggccggcCA...

Freiburg standard N-partPrefix is identical to the BioBrick prefix for coding sequences (EcoRI, NotI, XbaI, part in gray):

gaattccgcggccgcttctagATG...

Suffix

BioBrick 3.0 FusionSuffix (part in gray, AgeI, SpeI, NotI, PstI; original BioBrick suffix underlined):

...ACaccggttaatactagtagcggccgctgcag</b>

Combining the respective prefix and suffix generates the following FusionPart and N-part:

FusionPart

                          NaeI     BsrFI                    SfcI
 ApoI                  BsrFI |     BsaWI                 MspA1I|
EcoRI   NotI    XbaI  NgoMIV |      AgeI      SpeI    NotI    ||PstI
    |      |       |       | |         |         |       |    ||   |
    GAATTCgcggccgctTCTAGAtgGCCGGCnnnnnnACCGGTtaatACTAGTagcggccgCTGCAG
  1 ---------+---------+---------+---------+---------+---------+----- 65
    CTTAAGcgccggcgaAGATCTacCGGCCGnnnnnnTGGCCAattaTGATCAtcgccggcGACGTC
c     I  R  G  R  F  *  M  A  G  ?  ?  T  G  *  Y  *  *  R  P  L  Q   -

N-part

                             BsrFI                    SfcI
 ApoI                        BsaWI                 MspA1I|
EcoRI   NotI    XbaI          AgeI      SpeI    NotI    ||PstI
    |      |       |             |         |       |    ||   |
    GAATTCgcggccgctTCTAGAtgnnnnnnACCGGTtaatACTAGTagcggccgCTGCAG
  1 ---------+---------+---------+---------+---------+--------- 59
    CTTAAGcgccggcgaAGATCTacnnnnnnTGGCCAattaTGATCAtcgccggcGACGTC
c     I  R  G  R  F  *  M  ?  ?  T  G  *  Y  *  *  R  P  L  Q   -

Protocols

• See [http://openwetware.org/wiki/PrbbBB:Protocols Protocols for assembly of Freiburg parts] on OpenWetWare

References

• [http://hdl.handle.net/1721.1/45140 BBF RFC 25: Fusion Protein (Freiburg) Biobrick assembly standard] by Kristian M. Müller, Katja M. Arndt, the 2007 Freiburg iGEM team, and Raik Grünberg

<biblio>

1. Nishikubo pmid=16140408
2. Pfleger pmid=16247774
3. Varshavsky pmid=8901547

</biblio>

• [http://www.neb.com/nebecomm/products_Intl/productR0564.asp NgoMIV at NEB]