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| | how you used this part and how it worked out. | | how you used this part and how it worked out. |
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| | + | ===Usage and Biology=== |
| | ===Applications of BBa_K331030=== | | ===Applications of BBa_K331030=== |
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| − | In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks <html><a href="https://parts.igem.org/wiki/index.php/Part:BBa_K249005" target="new"><font color="#00DC00">BBa_K249005</font></a></html> and <html><a href="https://parts.igem.org/wiki/index.php/Part:BBa_K249004" target="new"><font color="#00DC00">BBa_K249004</font></a></html> respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (<html><a href="https://parts.igem.org/wiki/index.php/Part:BBa_K331023" target="new"><font color="#00DC00">BBa_K331023</font></a></html>) or N-terminus (<html><a href="https://parts.igem.org/wiki/index.php/Part:BBa_K331022" target="new"><font color="#00DC00">BBa_K331022</font></a></html>) (and preceded by a ribosomal binding site – <html><a href="https://parts.igem.org/wiki/index.php/Part:BBa_B0034" target="new"><font color="#00DC00">BBa_B0034</font></a></html>) were synthesized. We used our <html><a href="http://2010.igem.org/Team:Lethbridge/Notebook/Protocols#Assembly_of_BioBricks_using_the_Red.2FWhite_3-Antibiotic_Assembly_Method"><font color="#00DC00"> Red/White 3-Antibiotic assembly method</font></a></html> to add a tetracycline repressible promoter (<html><a href="https://parts.igem.org/wiki/index.php/Part:BBa_R0010" target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This addition generated BioBricks <html><a href="https://parts.igem.org/wiki/index.php/Part:BBa_K331031" target="new"><font color="#00DC00">BBa_K331031</font></a></html> and <html><a href="https://parts.igem.org/wiki/index.php/Part:BBa_K331030" target="new"><font color="#00DC00">BBa_K331030</font></a></html> for the C-terminal tagged and N-terminal tagged YFP respectively.
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| | ===User Reviews=== | | ===User Reviews=== |
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| − | ===Method===
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| − | The BioBrick containing plasmid was transformed into <i>Escherichia coli</i> DH5α cells. These cells were grown to an OD<sub>600</sub> of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.
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| − | This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5α), N-terminal tagged, and C-terminal tagged YFP were compared.
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| − | ===Results===
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| − | N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP.
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| − | <img src="https://static.igem.org/mediawiki/parts/3/3e/UofLNvsC-YFP1.jpg" width="450"/>
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| − | <img src="https://static.igem.org/mediawiki/parts/6/62/UofLNvsC-YFP2.jpg" width="450"/>
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| − | ===Conclusion===
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| − | Our results are consistent with the data reported by Bachmair <i>et al.</i> in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.
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| − | ===Reference===
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| − | <sup>1</sup>Bachmair A., Finley D., Varshavsky A. (1986), <b>In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue.</b> <i>Science</i> 234. <b>4773</b> 179-186.
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