Team:Lethbridge/Results
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The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein in vivo. | The placement of an oligoarginine sequence at the N-terminus of a protein will destabilize the protein in vivo. | ||
==<b><font color="white">Introduction</font></b>== | ==<b><font color="white">Introduction</font></b>== | ||
- | 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 research on the project, we came upon data suggesting that arginine residues at the N-terminus of a protein would significantly reduce its half-life, and causing degradation of our protein before it can be moved into the microcompartment. | + | 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 research on the project, we came upon data originally reported by Bachmair <i>et al.</i><sup>1</sup> suggesting that arginine residues at the N-terminus of a protein would significantly reduce its half-life, and causing 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. | We chose to investigate the how the placement of an oligoarginine sequence affects the stability of the protein to which it is fused. | ||
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==<b><font color="white">Method</font></b>== | ==<b><font color="white">Method</font></b>== | ||
In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <partinfo>BBa_K249005</partinfo> and <partinfo>K249004</partinfo> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<partinfo>BBa_K331023</partinfo>) or N-terminus (<partinfo>BBa_K331022</partinfo>) (and preceded by a ribosomal binding site – <partinfo>B0034</partinfo>) were synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<partinfo>BBa_R0010</partinfo>) for constitutive expression of the fusion protein. This addition generated BioBricks <partinfo>BBa_K331031</partinfo> and <partinfo>BBa_K331030</partinfo> for the C-terminal tagged and N-terminal tagged YFP respectively. | In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <partinfo>BBa_K249005</partinfo> and <partinfo>K249004</partinfo> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<partinfo>BBa_K331023</partinfo>) or N-terminus (<partinfo>BBa_K331022</partinfo>) (and preceded by a ribosomal binding site – <partinfo>B0034</partinfo>) were synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<partinfo>BBa_R0010</partinfo>) for constitutive expression of the fusion protein. This addition generated BioBricks <partinfo>BBa_K331031</partinfo> and <partinfo>BBa_K331030</partinfo> for the C-terminal tagged and N-terminal tagged YFP respectively. |