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Team:TUDelft/Recommendations
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After a great summer of hard work, we've learned quite a few things. The most important are listed below. | After a great summer of hard work, we've learned quite a few things. The most important are listed below. | ||
| - | ===Protein | + | ===Temperature sensitivity - Protein measurements=== |
Due to the fact it is impossible to state that lysis has a constant efficiency, it is not possible to take the cell density before lysis as a value for correcting luciferase activity. Therefore it is important to be able to measure something in the soluble fraction of your sample correlating to actual biomass . We decided to use a total protein content (Bicinchoninic acid assay) for the value to correct luciferase activity by. However, the lysis buffer (of unknown composition) in the luciferase assay kit appeared to interfere with our protein measurements. To prevent this interference we tried precipitating the protein in the samples, to get rid of the (unknown) interfering agent in the buffer, using 4 different methods. Although calibration curves first treated with lysis buffer and then precipitated were varying 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. | Due to the fact it is impossible to state that lysis has a constant efficiency, it is not possible to take the cell density before lysis as a value for correcting luciferase activity. Therefore it is important to be able to measure something in the soluble fraction of your sample correlating to actual biomass . We decided to use a total protein content (Bicinchoninic acid assay) for the value to correct luciferase activity by. However, the lysis buffer (of unknown composition) in the luciferase assay kit appeared to interfere with our protein measurements. To prevent this interference we tried precipitating the protein in the samples, to get rid of the (unknown) interfering agent in the buffer, using 4 different methods. Although calibration curves first treated with lysis buffer and then precipitated were varying 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 this problem of lysis buffer interference was trying various different lysis methods, including sonication, using glass beads in a fastprep or using lysozyme and glass sand in a beadbeater. Although it looked like all these methods can destroy some of the activity of luciferase in the sample, time was lacking to optimize each method and 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, although it is not known what the total protein of these samples is. | A different approach to solve this problem of lysis buffer interference was trying various different lysis methods, including sonication, using glass beads in a fastprep or using lysozyme and glass sand in a beadbeater. Although it looked like all these methods can destroy some of the activity of luciferase in the sample, time was lacking to optimize each method and 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, although it is not known what the total protein of these samples is. | ||
| - | ===Results with sonication=== | + | ===Temperature sensitivity - Results with sonication=== |
Using sonication of our samples, we obtained our most reliable results. These indicated, as displayed [https://2008.igem.org/Image:TUDelft221508a.png here], that our strain K115035 shows the temperature induced switch-like behavior which was the aim of our project. | Using sonication of our samples, we obtained our most reliable results. These indicated, as displayed [https://2008.igem.org/Image:TUDelft221508a.png here], that our strain K115035 shows the temperature induced switch-like behavior which was the aim of our project. | ||
| - | == | + | ===Color Pathway=== |
| + | It might have been a bit optimistic to expect it being possible to PCR 15 genes out of ''Escherichia coli'' and ''Saccharomyces cerevisiae'' in addition to making the temperature sensitive input system work (with only 2 wetlab guys). Still it was a good choice to split the project into two paths, as advised at the teachers workshop in Paris, as this allowed us to choose for the most promising path, should one of them fail. When it became obvious the actual color producing genes we aimed to get from the registry were of low quality (bad sequencing or did not grow at all) we gave priority to the temperature sensitive input. In the end we did make some progress towards constructing our envisioned color pathway by PCR-ing three of the genes out of ''E. coli'', which have been sent to the registry and to a sequencing company. | ||
| + | ==Future work== | ||
| + | In | ||
{{Template:TUDelftiGEM2008_sidebar}} | {{Template:TUDelftiGEM2008_sidebar}} | ||
Revision as of 10:30, 29 October 2008
Contents |
Conclusions & Recommendations
Conclusions
After a great summer of hard work, we've learned quite a few things. The most important are listed below.
Temperature sensitivity - Protein measurements
Due to the fact it is impossible to state that lysis has a constant efficiency, it is not possible to take the cell density before lysis as a value for correcting luciferase activity. Therefore it is important to be able to measure something in the soluble fraction of your sample correlating to actual biomass . We decided to use a total protein content (Bicinchoninic acid assay) for the value to correct luciferase activity by. However, the lysis buffer (of unknown composition) in the luciferase assay kit appeared to interfere with our protein measurements. To prevent this interference we tried precipitating the protein in the samples, to get rid of the (unknown) interfering agent in the buffer, using 4 different methods. Although calibration curves first treated with lysis buffer and then precipitated were varying 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 this problem of lysis buffer interference was trying various different lysis methods, including sonication, using glass beads in a fastprep or using lysozyme and glass sand in a beadbeater. Although it looked like all these methods can destroy some of the activity of luciferase in the sample, time was lacking to optimize each method and 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, although it is not known what the total protein of these samples is.
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.
Color Pathway
It might have been a bit optimistic to expect it being possible to PCR 15 genes out of Escherichia coli and Saccharomyces cerevisiae in addition to making the temperature sensitive input system work (with only 2 wetlab guys). Still it was a good choice to split the project into two paths, as advised at the teachers workshop in Paris, as this allowed us to choose for the most promising path, should one of them fail. When it became obvious the actual color producing genes we aimed to get from the registry were of low quality (bad sequencing or did not grow at all) we gave priority to the temperature sensitive input. In the end we did make some progress towards constructing our envisioned color pathway by PCR-ing three of the genes out of E. coli, which have been sent to the registry and to a sequencing company.
Future work
In
