Regulatory

Part:BBa_K873002:Experience

Designed by: yue hu   Group: iGEM12_SEU_A   (2012-09-18)


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Characterization

WashU_StLouis

The WashU StLouis iGEM team measured the strength of this HSP by attaching it to a GFP and measuring florescence at various temperatures.

Protocol:

  1. Combined biobrick with a GFP biobrick BBa_E0840 to make a measurable device
  2. Grew biobrick, and DH10B as a control, overnight in LB and antibiotic overnight
  3. Measured the OD600 of each sample and diluted it down to OD600=0.25
  4. Grew diluted cultures for two hours in M9 minimal media and antibiotic
  5. Set up two plates, each with three samples of each culture
  6. Set up one plate in plate reader at 42C and measured absorbance and fluorescence every 15 minutes until the growth leveled off. During this time, another plate was in 37C shaker and was measured at the beginning and end
  7. Next day, repeated protocol but set the plate in the plate reader to 30C

The plate in the plate reader was measured every fifteen minutes until the growth of the cells leveled out. This is observed when the absorbance of the cells stayed the same for multiple cycles. The fluorescence for the 30C and 42C plates were normalized and plotted over time. As seen in Figure 1 below, there was a spike in fluorescence shortly after the HSP was activated on the 42C plate. On the other hand, the HSP on the 30C plate was never activated. For both, fluorescence decreased greatly over time. At this time, we do not know if this is due to the HSP or the GFP device. The decrease in fluorescence over time may be due to photobleaching as well.

Figure 1

Figure 2 below shows the measured fluorescence for each temperature before and after the experiment. Labels are given for Test 1 and Test 2 in order to differentiate between the two days the experiment was run. As expected, there is little difference between the temperatures before the experiment was run, but even after the HSP was heat activated, there was no discernible difference between the fluorescence of the various temperatures.

In conclusion, we saw that this HSP biobrick was not activated by high temperatures.

Figure 2

In order to reaffirm these results, the Cardiff University iGEM team repeated this protocol and got very similar results, as seen in Figure 3 and Figure 4.

Figure 3
Figure 4


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