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Revision as of 20:59, 21 October 2007

Proposal for a BioBrick extension for fusion proteins

iGEM team Freiburg

Introduction

The BioBrick prefix and suffix is an excellent and universal way to combine various parts, e.g. promoter region, gene of interest, terminator etc. However, the generation of fusion proteins is not possible due to the generation of a stop codon at the SpeI/Xba 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...

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

Consequently, fusion proteins can so far only be designed as one part but not in a modular fashion. Therefore, we developed a new generation of BioBricks completely compatible with the first version but with two additional compatible restriction sites to allow for the modular construction of protein fusion parts. 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. We call this new version BioBricks 2.0.

Proposal

iGEM parts for mix-and-match construction of fusion

proposed by Katja M. Arndt, Kristian M. Müller, University of Freiburg

• Both parts are compatible with all standard iGEM parts as they have the BioBricks prefix for coding sequences and the standard BioBrick suffix.
• nnnnnn is a place holder for the coding sequence of the respective part.
• 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 N-part is designed to be the start of a fusion protein or a stand-alone protein part, to be cloned via XbaI/PstI to any iGEM RBS expression part.
• The FusionPart is designed to 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 fused to the N-part.
• The FusionPart can also be a stand-alone part as it has a start codon after the XbaI site (BioBrick prefix for coding sequence), however two additional amino acids (A, G) are encoded before the start of the protein.

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   -

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   -

Discussion

References