Part:BBa_K331031:Experience

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Applications of BBa_K331031

The long term goal of our team is to utilize an oligoarginine tail to specifically target enzymes into a microcompartment composed of modified lumazine synthase subunits. While conducting background research on the project, we came upon data originally reported by Bachmair et al.¹ suggesting that the identity of the amino acid at the N-terminus of a protein is related to its half-life, and mostly notably, that arginine residues at the are destabilizing. This data suggests that by placing an arginine at the N-terminus of a protein to be targeted into a lumazine synthase microcompartment would cause degradation of our protein before it can be moved into the microcompartment.

We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused.

In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks BBa_K249005 and BBa_K249004 respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (BBa_K331023) or N-terminus (BBa_K331022) (and preceded by a ribosomal binding site – BBa_B0034) were synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (BBa_R0040) for constitutive expression of the fusion protein. This addition generated BioBricks BBa_K331031 and BBa_K331030 for the C-terminal tagged and N-terminal tagged YFP respectively.

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