Latest revision as of 23:45, 25 October 2007

Proposal for a BioBrick extension for fusion proteins

iGEM team Freiburg

Introduction

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, also proteins consist of different parts and thus the "mix-and-match" combination of fusion proteins is an important task in Synthetic Biology. However, this modular assembly from BioBrick parts is currently 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...

encoding 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 BioBrick 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 3.0.

Proposal

We propose to extend the standard 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. A combination of two such FusionParts 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 Methionin 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 factors of the BioBrick 3.0 design

Strategy for iGEM BioBrick 3.0 parts for mix-and-match construction of fusion proteins

developed by the iGEM Team Freiburg

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

BioBrick 3.0 FusionPrefix (EcoRI, NotI, XbaI, NgoMIV, part in gray;

original BioBrick prefix for coding sequences underlined):

gaattccgcggccgcttctagatggccggcCA...

BioBrick 3.0 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

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   -

Discussion

References

NgoMIV NEB information
Nishikubo T, Nakagawa N, Kuramitsu S, Masui R "Improved heterologous gene expression in Escherichia coli by optimization of the AT-content of codons immediately downstream of the initiation codon." J Biotechnol. 2005 Dec 6;120(4):341-6
Pfleger BF, Fawzi NJ, Keasling JD "Optimization of DsRed production in Escherichia coli: effect of ribosome binding site sequestration on translation efficiency." Biotechnol Bioeng. 2005 Dec 5;92(5):553-8
Varshavsky A "The N-end rule: functions, mysteries, uses." Proc Natl Acad Sci U S A. 1996 Oct 29;93(22):12142-9 [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=8901547&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum pubmed]

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