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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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| | ===User Reviews=== | | ===User Reviews=== |
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| − | ===Method=== | + | ===Usage and Biology=== |
| − | 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>.
| + | ===Applications of BBa_K331033=== |
| − | <br><br>
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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=== | + | |
| − | 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:UofLcfpfigureblack.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=== | + | |
| − | <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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| − | <sup>2</sup>McRae S.R., Brown C.L., Bushell G.R. (2005), Rapid Purification of EGFP, EYFP, and ECFP with high yield and purity. <i>Protein Expression and Purification</i> 41. 1 121-127.
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Latest revision as of 00:11, 29 October 2010
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Usage and Biology
Applications of BBa_K331033
