Difference between revisions of "Part:BBa I13522:Experience"
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<p> Stock solutions of GFP 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 GFP 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> | ||
| − | <div class="imgbox" style="width: | + | <div class="imgbox" style="width:720px;margin: 0 auto;"> |
| − | <img class="border" src="https://static.igem.org/mediawiki/ | + | <img class="border" src="https://static.igem.org/mediawiki/2011/f/f8/ICL_Heat_Denaturation_Curve_of_Fluorescent_Proteins.png" width=700px /> |
| + | <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> | ||
| + | </div> | ||
| − | + | 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): | |
| + | <br> | ||
| + | <img src= "https://static.igem.org/mediawiki/2011/0/0d/ICL_Equation_to_find_PTm50.png" width="600px"/> | ||
</html> | </html> | ||
Revision as of 13:36, 21 September 2011
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.
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.
The characterization was performed by METU-TURKEY IGEM 2010 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 GFP 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.
This characterisation was performed by the Imperial College London 2011 Team.
Applications of BBa_I13522
User Reviews
UNIQ91df09da617b5e1b-partinfo-00000002-QINU
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Antiquity |
This review comes from the old result system and indicates that this part did not work in some test. |
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