Thermoinduction system activity variation with temperature

Background

We are using the thermoinduction promoter system [http://partsregistry.org/wiki/index.php/Part:BBa_K098995 BBa_K098995] to activate module 3 (genome deletion) when the temperature is raised to 42 degrees.

We have ligated the thermoinduction promoter system [http://partsregistry.org/wiki/index.php/Part:BBa_K098995 BBa_K098995] to the GFP reporter to form our own testing construct [http://partsregistry.org/wiki/index.php/Part:BBa_K200022 BBa_K200022] . This allows us to follow thermoinduction of the promoter by changes in GFP fluorescence.

Aim

To investigate the behaviour of the lamda-cI thermoinducible promoter and show repression at the low temperature of 28°C and activation when the temperature is raised to 42°C.

Experimental method

Cells were grown at the 2 different temperatures of 28°C and 42°C. There was in addition a set of cells that were shifted from 28°C to 42°C so that we could characterise the change in GFP fluorescence during the transition between temperatures.

In order to characterize the thermoinducible promoter properly, we have used 2 sets of control cells:

• 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. These serve as cells without any fluorescence.

Results

Variation in the blank

The level of variation in the blank absorbance and fluorescence is within a narrow range. However, we cannot immediately assume that it is constant, given that we are dealing with relatively low baseline values.

In order to take into account this variation, the growth rate and fluorescence output of the curves was computed for different values within the range, and the purpose was to decide if this variation had a significant impact.

Click below for more information on blank variation analysis:

Absorbance blank variation

Fluorescence blank variation

The level of variation in the blank absorbance and fluorescence is within a narrow range. However, we cannot immediately assume that it is constant, given that we are dealing with relatively low baseline values. (0.05-0.09)
Figure 3: Variation in absorbance of the blank well over time at 28 ºC


Figure 4: Variation in absorbance of the blank well over time at 42 ºC

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.

Absorbance data

Understanding of the fluorescence data requires normalization with cell growth data, in the form of optical density (absorbance).

28 degrees Celsius

Figure 1: Absorbance at 28 degrees Celsius

42 degrees Celsius

Figure 2: Absorbance at 42 degrees Celsius
Plotting raw absorbance data on its own does not provide sufficient information about the effects of temperature on our system. Therefore, further analysis is required. This includes recording the variation in absorbance of the blank well (containing only the M9 medium) and normalizing absorbance data against the latter to spot ay possible trends in growth rate variations at different temperatures.

Variation in absorbance of the blank

Figure 3 shows that the level of variation in the blank absorbance is within a narrow range (0.05-0.09). However, we cannot immediately assume that it is constant, given that we are dealing with relatively low Optical density values.
Figure 3: Variation in absorbance of the blank well over time at 28 ºC
In order to take into account this variation, the growth rate of the curves was computed for different values within the range, and the purpose was to decide if this variation had a significant impact on growth rate.

Figure 4: Variation in absorbance of the blank well over time at 42 ºC

Calculation of growth rate of the population for varying blank levels

28 ºC

• Growth rate variation is low for different blank values(0.004-0.005)
• See figure 5 for an example.
• The growth rate of the culture seems to be constant at the values mentioned (0.004 to 0.005 /min) but the positive control seems to be higher. We believe that this variation is due to noise.

42 ºC

• The linear trend in variation of the blank can be explained due to evaporation effects. In this case, the variation was accounted for directly, so it was unnecessary to evaluate the growth rate for different blank values.
• The y-intercept in the plot is not reliable but the trend is apparent.
• We can observe exponential growth for the first 100 minutes (figure 6) at a rate of roughly 0.012 /min.

Conclusion

Cells grow faster at 42 ºC than at 28 ºC.

Analysis