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Team:TUDelft/Temperature conclusions
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===Temperature sensitivity - Protein measurements=== | ===Temperature sensitivity - Protein measurements=== | ||
| - | + | Because lysis efficiency seems to vary during and in between experiments, it is not possible to take the cell density before lysis as a value for correcting luciferase activity. Protein content measurements of the soluble fraction after lysis should be conducted to reliably normalize luminescence. Total protein content determination was performed by Bicinchoninic acid assay (BC assay). However, the lysis buffer (of unknown composition) in the luciferase assay kit interfered with the protein measurements. To circumvent this interference we tried precipitating the protein in the samples using 4 different methods to get rid of the (unknown) interfering agent in the lysis buffer. Although calibration curves that were first treated with lysis buffer and then precipitated varied from quite linear (R<sup>2</sup>=0.983) to not linear at all (R<sup>2</sup><0), samples containing the soluble fraction of lysed cells usually gave protein contents of below 0 mg/ml. | |
| - | A different approach to solve | + | A different approach to solve the problem of lysis buffer interference was trying different lysis methods. Sonication, using glass beads in a FastPrep or using lysozyme and glass sand in a bead beater. Some luciferase activity was lost with all these methods, and time was lacking to optimize each method. The sonication protocol, although time consuming, seemed most reliable in our experiments. Still, this method is not optimal, as samples lysed with lysis buffer show a lot higher raw luciferase output. |
===Temperature sensitivity - Results with sonication=== | ===Temperature sensitivity - Results with sonication=== | ||
Revision as of 17:44, 29 October 2008
Conclusions Temperature
After a great summer of hard work, we've learned quite a few things. The most important about the thermometer RNA are listed below.
Temperature sensitivity - Protein measurements
Because lysis efficiency seems to vary during and in between experiments, it is not possible to take the cell density before lysis as a value for correcting luciferase activity. Protein content measurements of the soluble fraction after lysis should be conducted to reliably normalize luminescence. Total protein content determination was performed by Bicinchoninic acid assay (BC assay). However, the lysis buffer (of unknown composition) in the luciferase assay kit interfered with the protein measurements. To circumvent this interference we tried precipitating the protein in the samples using 4 different methods to get rid of the (unknown) interfering agent in the lysis buffer. Although calibration curves that were first treated with lysis buffer and then precipitated varied from quite linear (R2=0.983) to not linear at all (R2<0), samples containing the soluble fraction of lysed cells usually gave protein contents of below 0 mg/ml.
A different approach to solve the problem of lysis buffer interference was trying different lysis methods. Sonication, using glass beads in a FastPrep or using lysozyme and glass sand in a bead beater. Some luciferase activity was lost with all these methods, and time was lacking to optimize each method. The sonication protocol, although time consuming, seemed most reliable in our experiments. Still, this method is not optimal, as samples lysed with lysis buffer show a lot higher raw luciferase output.
Temperature sensitivity - Results with sonication
Using sonication of our samples, we obtained our most reliable results. These indicated, as displayed here, that our strain K115035 shows the temperature induced switch-like behavior which was the aim of our project.
