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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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| − | ===Applications of BBa_K331033===
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| − | ===Introduction===
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| − | Prior to introducing a oligoarginine tail onto the <html><a href="http://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00">catechol-2,3-dioxygenase</font></a></html> protein with the intention of directing the fusion protein into a lumazine synthase microcompartment, there must be some confirmation that oligoarginine tail is indeed causing the protein to which it is attached to localize within the microcompartment. We chose to utilize a technique known as fluorescent resonance energy transfer<sup>1</sup> (FRET) to verify localization. In FRET, when a dye pair comes into close proximity to one another, one dye (the donor) will transfer some of its energy to the second dye (the acceptor). This will cause the acceptor to fluoresce without direct excitation. The dye pair we have chose is CFP as the donor and yellow fluorescent protein (YFP) as the acceptor.
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| − | Here we investigate the ability of <html><a href="https://parts.igem.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> to produce CFP and emit light at its characteristic emission wavelength.
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| | ===User Reviews=== | | ===User Reviews=== |
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| − | ===Method===
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| − | In order to characterize the tetracycline repressible CFP (BioBrick <html><a href="https://parts.igem.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>), cyan fluorescent protein (CFP-<html><a href="https://parts.igem.org/wiki/index.php/Part:BBa_E0020"target="new"><font color="#00DC00"">BBa_E0020</font></a></html>) with an oligoarginine tag fused to the C-terminus (<html><a href="https://parts.igem.org/wiki/index.php/Part:BBa_K331025"target="new"><font color="#00DC00">BBa_K331025</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>) was synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (<html><a href="https://parts.igem.org/wiki/index.php/Part:BBa_”R0040"target="new"><font color="#00DC00">BBa_R0040</font></a></html>) for constitutive expression of the fusion protein. This construction yielded BioBrick <html><a href="https://parts.igem.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html>.
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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 in LB media, and diluted 1:10 with MilliQ H<sub>2</sub>O immediately prior to analysis by fluorescent spectroscopy.
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| − | <br><br>
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| − | This dilution of cells was excited at 439 nm, and the emission spectra was read from 444 nm to 650 nm.
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| − | <br><br>
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| − | ===Results===
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| − | Fluorescence at 476 nm was observed. This peak, along with a shoulder occurring at approximately 510 nm is consistent with results obtained by McRae<sup>1</sup> <i>et al.</i> in their rapid purification of ECFP.<br>
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| − | [[image:UofLcfpfigurewhite.jpg|450px|center]]
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| − | <br>
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| − | ===Conclusion===
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| − | The BioBrick <html><a href="https://parts.igem.org/wiki/index.php/Part:BBa_K331033" target="new"><font color="#00DC00">BBa_K331033</font></a></html> that we constructed generates CFP in the absence of tetracycline, as expected.
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| − | <br><br>
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| − | ===Reference===
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| − | <sup>1</sup>Förster T. (1948), Zwischenmolekulare Energiewanderung und Fluoreszenz. <i>Annalen der Physik</i>, 437: 55-75.
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| − | <br><br>
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Revision as of 01:45, 28 October 2010
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