Team:Carnegie Mellon/mRFP1

From 2013.igem.org

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<h2>mRFP1 Characterization</h2>
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<h2>mRFP1 characterization</h2>
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[[Image:CRQ-mRFP1.png|thumb|600px|center|The QYG-derived chromophore of the mRFP1 protein. (Z)-2-(2-(4-amino-1-imino-4-oxobutyl)-4-(4-hydroxybenzylidene)-5-oxo-4,5-dihydro-1H-imidazol-1-yl)acetic acid. The extended π-system is highlighted in red.]]
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[[Image:CRQ-mRFP1.png|thumb|300px|center|The QYG-derived chromophore of the mRFP1 protein. (Z)-2-(2-(4-amino-1-imino-4-oxobutyl)-4-(4-hydroxybenzylidene)-5-oxo-4,5-dihydro-1H-imidazol-1-yl)acetic acid. The extended π-system is highlighted in red.]]
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<p align="center"><b> Characterization of the Photostability of mRFP1</b></p></html>
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[[Image:MRFP1.png|center|thumb|400px|Photobleaching curve of mRFP1 with a HBO100 mercury-arc lamp]]
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XL10 Ultracompetent cells were transformed with <partinfo>BBa_E1010</partinfo> cloned with <partinfo>BBa_B0034</partinfo> as the RBS and  <partinfo>BBa_R0010</partinfo> as the wild-type lac promoter and induced overnight with IPTG.The overnight was bleached for 180 minutes with HBO100 (100W Mercury-arc lamp). Fluorescence data was taken using a Tecan Safire II with the parameters shown in Table 3. Fluorescence values are shown in Table 4. <html><br />
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<table cellpadding="2" border="1px" cellspacing="0" align="center" width="40%">
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<caption><p align="justify"><b>Table 3</b> Tecan Safire II Parameters</p></caption>
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<tr><td><b>Excitation (nm)</b></td><td>585</td></tr>
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<tr><td><b>Emission (nm)</b></td><td>610</td></tr>
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<tr><td><b>Excitation bandwidth (nm)</b></td><td>10</td></tr>
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<tr><td><b>Emission bandwidth (nm)</b></td><td>10</td></tr>
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<tr><td><b>Gain</b></td><td>63</td></tr>
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<tr><td><b>Number of reads</b></td><td>10</td></tr>
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<tr><td><b>Integration Time (microseconds)</b></td><td>40</td></tr>
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<table cellpadding="2" border="1px" cellspacing="0" align="center" width="40%">
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<caption><p align="justify"><b>Table 4</b> Shows the fluorescence data over time during photobleaching.</p></caption>
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<tr><td align="center"><b>Time (minutes)</b></td><td align="center"><b>Fluorescence (RFU)</b></td>
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<tr><td align="left">0</td><td align="right">48694</td></tr>
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<tr><td align="left">20</td><td align="right">43083</td></tr>
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<tr><td align="left">40</td><td align="right">36842</td></tr>
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<tr><td align="left">60</td><td align="right">30239</td></tr>
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<tr><td align="left">80</td><td align="right">31281</td></tr>
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<tr><td align="left">100</td><td align="right">25273</td></tr>
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<tr><td align="left">120</td><td align="right">21467</td></tr>
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<tr><td align="left">140</td><td align="right">18081</td></tr>
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<tr><td align="left">160</td><td align="right">15251</td></tr>
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<tr><td align="left">180</td><td align="right">14427</td></tr>
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<h2>References</h2>
<h2>References</h2>
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Campbell,R.E., Tour,O., Palmer,A.E., Steinbach,P.A., Baird,G.S., Zacharias,D.A. and Tsien,R.Y. <i>A monomeric red fluorescent protein</i>. Proc. Natl. Acad. Sci. U.S.A. 99 (12), 7877-7882 (2002)
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Latest revision as of 01:21, 28 September 2013

Killer Red




mRFP1 characterization

The QYG-derived chromophore of the mRFP1 protein. (Z)-2-(2-(4-amino-1-imino-4-oxobutyl)-4-(4-hydroxybenzylidene)-5-oxo-4,5-dihydro-1H-imidazol-1-yl)acetic acid. The extended π-system is highlighted in red.

Characterization of the Photostability of mRFP1

Photobleaching curve of mRFP1 with a HBO100 mercury-arc lamp

XL10 Ultracompetent cells were transformed with <partinfo>BBa_E1010</partinfo> cloned with <partinfo>BBa_B0034</partinfo> as the RBS and <partinfo>BBa_R0010</partinfo> as the wild-type lac promoter and induced overnight with IPTG.The overnight was bleached for 180 minutes with HBO100 (100W Mercury-arc lamp). Fluorescence data was taken using a Tecan Safire II with the parameters shown in Table 3. Fluorescence values are shown in Table 4.

Table 3 Tecan Safire II Parameters
Excitation (nm)585
Emission (nm)610
Excitation bandwidth (nm)10
Emission bandwidth (nm)10
Gain63
Number of reads10
Integration Time (microseconds)40

Table 4 Shows the fluorescence data over time during photobleaching.
Time (minutes)Fluorescence (RFU)
048694
2043083
4036842
6030239
8031281
10025273
12021467
14018081
16015251
18014427

References

Campbell,R.E., Tour,O., Palmer,A.E., Steinbach,P.A., Baird,G.S., Zacharias,D.A. and Tsien,R.Y. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. U.S.A. 99 (12), 7877-7882 (2002)

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