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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

<p>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

The following list summarizes the most important factors of the BioBrick 2.0 design

Strategy for iGEM BioBrick 2.0 parts for mix-and-match construction of fusion proteins

developed by Katja M. Arndt, Kristian M. Müller, University of 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

Suffix

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

FusionPart

N-part

Discussion

References