Difference between revisions of "Part:BBa I13521:Experience"
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<p>Stock solutions of mRFP were prepared by extracting the protein from cell lysate, and then 50μl aliquots of the solution were heated in a PCR thermocycler along a temperature gradient.</p>
 
<p>Stock solutions of mRFP were prepared by extracting the protein from cell lysate, and then 50μl aliquots of the solution were heated in a PCR thermocycler along a temperature gradient.</p>
 
<p>After two hours, 30μl was removed from each aliquot and diluted with 170μl of 20mM Tris buffer to give 200μl samples. The samples were then measured by fluorescence on a 96-well plate. The corresponding curve was plotted on the graph below.</p>
 
<p>After two hours, 30μl was removed from each aliquot and diluted with 170μl of 20mM Tris buffer to give 200μl samples. The samples were then measured by fluorescence on a 96-well plate. The corresponding curve was plotted on the graph below.</p>
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<p><i>Fig. 4: Results of the heat denaturation experiment. The temperature at which half of the protein is denatured measured by looking at its fluorescence (PTm50) mRFP1: 92.3°C; GFPmut3b: 59.1°C; Dendra2: 83.7°C; sfGFP: 75.3°C.</i></p>
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The sigmoidal curves that were calculated gave us the following function in order to find K which we also call PTm50 (temperature at which half of the protein is denatured measured by looking at its fluorescence):
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<img src= "https://static.igem.org/mediawiki/2011/0/0d/ICL_Equation_to_find_PTm50.png" width="600px"/>
 
<p>This shows that the protein denatures at around 81°C</p>.  
 
<p>This shows that the protein denatures at around 81°C</p>.  
 
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Revision as of 13:35, 21 September 2011

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_I13521



For parts BBa_I13521 and BBa_I13522, their excitation and emission maxima were determined. BBa_B0034 was used as a negative control. These data were used later in following characterization experiments as set parameters. The host was E. coli Top Ten in all experiments.

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The graph above shows the emission scan for RFP in part BBa_I13521. Since it was the best option in terms of fluorescence yield, 570 nm was set as the excitation wavelength. As it can be seen from the graph, maximum emission wavelength is 605 nm.
Meanwhile, the graph below shows the emission scan for GFP in part BBa_I13522. Since it was the best option in terms of fluorescence yield, 395 nm was set as the excitation wavelength. As it can be seen from the graph, maximum emission wavelength is 515 nm.

For the parts BBa_I13521 and BBa_I13522 which are mentioned above, confocal laser scanning microscope images were taken in the host Top Ten.
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Characterization was performed by METU-TURKEY IGEM2010 Team

Thermostability Assay

For this BioBrick, the denaturation temperature was determined by heating the protein at a range of temperatures, and then measuring the fluorescence. This is a useful characterisation as it allows the selection of an appropriate reporter gene for the required temperature.

Stock solutions of mRFP were prepared by extracting the protein from cell lysate, and then 50μl aliquots of the solution were heated in a PCR thermocycler along a temperature gradient.

After two hours, 30μl was removed from each aliquot and diluted with 170μl of 20mM Tris buffer to give 200μl samples. The samples were then measured by fluorescence on a 96-well plate. The corresponding curve was plotted on the graph below.

Fig. 4: Results of the heat denaturation experiment. The temperature at which half of the protein is denatured measured by looking at its fluorescence (PTm50) mRFP1: 92.3°C; GFPmut3b: 59.1°C; Dendra2: 83.7°C; sfGFP: 75.3°C.

The sigmoidal curves that were calculated gave us the following function in order to find K which we also call PTm50 (temperature at which half of the protein is denatured measured by looking at its fluorescence):

This shows that the protein denatures at around 81°C

.

This characterisation was performed by the Imperial College London 2011 Team.

User Reviews

UNIQ9bffd0b9e6543e9b-partinfo-00000003-QINU

•••••

melissali

Very visible to the naked eye (vs. CFP, YFP were not very visible without UV excitation).

UNIQ9bffd0b9e6543e9b-partinfo-00000005-QINU