Experiment Rationale

To investigate the behaviour of the lamda-cI thermoinducible promoter and show that when temperature is low (at 28 degrees Celsius), there is low fluorescence output. This shows that the genome deletion module is repressed. When temperature is raised to 42 degrees Celsius, fluorescence increases, indicating that the repression is lifted. We are looking at both absorbance and fluorescence data. This analysis serves to characterize the construct [http://partsregistry.org/wiki/index.php/Part:BBa_K200022 BBa_K200022], submitted by Harvard last year.

Summary of method

In order to characterize the thermoinducible promoter, absorbance (optical density) and fluoresence data were recorded over time for:

• Cells containing the BBa_K200022 construct: The thermoinducible promoter.
• Positive control cells: Containing the [http://partsregistry.org/Part:BBa_I13522 BBa_I13522], acting as a baseline comparison by constitutively expressing GFP.
• Negative control cells: These contain the thermoinducible promoter on its own ( [http://partsregistry.org/Part:BBa_K098995 BBa_K098995]) with no GFP attached to it.

Analysis

Variation of the blank

Here we repeat a similar analysis to the previous part, except that this time we will account for variations in the blank for fluorescence data. However, we suspect that there has been a systematic error in the results returned by the fluorometer. The analysis brings out the data's most important features and compensates for these. All the raw data files will also be uploaded in the wiki for further details and explanations.

28 ºC

• At this temperature, variations of the blank levels have been taken to be between 250 and 300 Fluorescent units (discarding overshoots).
• This means that once again, fluorescence data must be normalized against different blank data values to account for this variation.

Figure 1: Fluorescence variation in blank wells at 28ºC

42 ºC

• Here, variations seem to be linear, as seen in the absorbance case so one of the causes for this may be evaporation.
• However, they may also be due to systematic error of the fluorometer.

Figure 2: Fluorescence variation in blank wells at 42ºC

Variation in fluorescence

28ºC

• Table 1 shows the raw values for fluorescence. Again, this was done for different blank values (for more details see the raw data file)

Table 1: Fluorescence results over time at 28 ºC

• At 28 ºC, we can clearly see that the fluorescence is repressed, relative to the constitutive promoter (positive control).
• The blue line is low, and fluorescence output is low. Therefore, the promoter is repressing downstream genes.

Figure 3: Fluorescence at 28 ºC

42ºC

Table 2: Fluorescence results at 42ºC

• In figure we can observe that fluorescence levels drift towards a steady state and are no longer low (as in the 28 ºC case).
• This plot starts at a value of 4000 fluorescence units because the cultures were shifted from 28 ºC to 42 ºC overnight.
• In figure 5 we have accounted for the effects of evaporation (as it happens due to an increase in temperature) so we can now see the positive control at a relatively constant level. This was not the case in figure 4, where evaporation was not taken into account. This, as mentioned earlier, could also be the reason of variation in the blank results.
• There is a clear difference with teh 28 ºC case, showing that indeed, at higher temperatures the downstream genes from the promoter are no longer repressed.

Figure 4: Fluorescence results at 42 degrees

Figure 5: Fluorescence results at 42 degrees (corrected for effect of evaporation)

Conclusion

The characterization of the thermoinducible promoter is as follows:

Hence, we can conclude that in the E.ncapsulator system, an increase in temperature de-represses the expression of downstream genes, which trigger the genome deletion phase.