Team:BCCS-Bristol/Bioscaffold/Protein Fusion Problem


iGEM 2009

Protein Fusion Problems

Each gene that encodes a protein has to have two stop codons at its 3' end (5'-TAATAA-3'). Moreover when two genes are fused together an additional third stop codon is created within the DNA scar (DNA region that connects the two genes). Hence this would prevent the expression of the second partner in the fusion protein.

Existing Solutions

The current iGEM assembly standards do not fully support protein fusions.

Current assembly standards address this issue poorly. Assembly standard 23 removes a base pair from the prefix and suffix respectively giving a 5'-ACT AGA-3' scar, which replaces the DNA scar stop codon but instead inserts a problematic Arginine residue that is highly positively charged and also not preferred by the E.coli' translational machinery. Assembly standard 25 adds an extra restriction consensus prior to the start codon of the upstream protein and this would upset the 5-6 base pair spacer rule that should exist between the RBS and the protein to ensure that translation is optimal. Neither standards address the problem with the two TAA TAA stop codons present in the biobrick sequence.

Only assembly standard 15, a concept known as Bioscaffold, tries to address this problem in some depth. The Bioscaffold is a DNA sequence that is ligated between the two protein encoding genes and contains restriction sites of atypical Type II restriction enzymes. The enzymes will cleave outside their recognition sequence hence removing the stop codons of the first protein in the fusion.

However the current scaffolds are not compatible as biobricks or not available in biobrick format and some require further manipulation with PCR primers.

A summary of canditate solutions to the protein fusion problem and how applying them may cause additional problems and considerations.

General concept of how the bioscaffold works