Difference between revisions of "Part:BBa K911001:Experience"
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We repeated this experiment, using a different range of magnesium concentrations (1μM - 5mM) and doing duplicates in alternate rows. The cells grew well, although they did not appear to do so in an exponential fashion. It is believed that this may be a feature of the minimal medium in which we were performing the assay, as failed tests using a rich defined medium (which, unfortunately, autofluoresced at GFP wavelengths due to the aromatic amino acids it contained) showed normal exponential growth with the same construct and at identical magnesium concentrations. | We repeated this experiment, using a different range of magnesium concentrations (1μM - 5mM) and doing duplicates in alternate rows. The cells grew well, although they did not appear to do so in an exponential fashion. It is believed that this may be a feature of the minimal medium in which we were performing the assay, as failed tests using a rich defined medium (which, unfortunately, autofluoresced at GFP wavelengths due to the aromatic amino acids it contained) showed normal exponential growth with the same construct and at identical magnesium concentrations. | ||
| − | Having collected OD620 data as well as fluorescence data, we hoped to generate graphs containing normalized | + | Having collected OD620 data as well as fluorescence data, we hoped to generate graphs containing fluorescence readings normalized to cell density. We used the following formula to normalize our data: |
| + | |||
| + | [[Image:NormalisationFormula.png|center]] | ||
| + | |||
| + | Initial analysis produced fairly convincing data | ||
We then attempted to transform the construct into Bacillus subtilis, with a view to repeat the same assay in this new chassis. Because the part originally comes from bacillus, we hoped that this might give more useful data. Unfortunately, we did not have time to characterise the part in this chassis, as our transformation attempts failed. This may be an excellent starting point for any future teams seeking to explore this part. | We then attempted to transform the construct into Bacillus subtilis, with a view to repeat the same assay in this new chassis. Because the part originally comes from bacillus, we hoped that this might give more useful data. Unfortunately, we did not have time to characterise the part in this chassis, as our transformation attempts failed. This may be an excellent starting point for any future teams seeking to explore this part. | ||
Revision as of 17:22, 26 September 2012
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how you used this part and how it worked out.
Applications of BBa_K911001
BBa_K911001 has been inserted into the following construct:
The sequence of this construct has been verified. E.coli colonies containing this construct expressed sfGFP, which was expected due to the leakiness of the lacI repressor, as well as the fact that the LB on which the cells were grown will have contained magnesium.
In order to test it, we attempted to induce GFP expression in transformed e.coli cells with both increasing magnesium concentrations and increasing IPTG concentrations. The cells were then interrogated at the correct GFP absorbtion and emission wavelengths (450nm and 545nm respectively) in a plate reader.
The results of our initial assay are shown below.
As can be seen, the assay did not give the expected results. At differing IPTG concentrations, the response to magnesium seems to have inverted.
We repeated this experiment, using a different range of magnesium concentrations (1μM - 5mM) and doing duplicates in alternate rows. The cells grew well, although they did not appear to do so in an exponential fashion. It is believed that this may be a feature of the minimal medium in which we were performing the assay, as failed tests using a rich defined medium (which, unfortunately, autofluoresced at GFP wavelengths due to the aromatic amino acids it contained) showed normal exponential growth with the same construct and at identical magnesium concentrations.
Having collected OD620 data as well as fluorescence data, we hoped to generate graphs containing fluorescence readings normalized to cell density. We used the following formula to normalize our data:
Initial analysis produced fairly convincing data
We then attempted to transform the construct into Bacillus subtilis, with a view to repeat the same assay in this new chassis. Because the part originally comes from bacillus, we hoped that this might give more useful data. Unfortunately, we did not have time to characterise the part in this chassis, as our transformation attempts failed. This may be an excellent starting point for any future teams seeking to explore this part.
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