After the integrated human practice work, we updated SynRT Toolkit to version 2.0. In version 2.0, we added 22 heat-repressible RNA-based thermosensors to Toolkit.
Heat-repressible RNA-based thermosensors were based on the RNase E. We measured these thermosensors' activity by using measurement device like before. But this time, we designed a new negative control. In the following, it will be called negative control-2. Negative control-2 has a cleavage site of RNase E. It will always be digested by enzyme. Due to the effiency of RNase E, we decided to use negative control-2 instead of traditional negative control.
We designed 100 heat-repressible RNA-based thermosensors, their sequences are different. After measurement, we removed some devices which show less sensing in temperature or have undesirable results, then we finally selected 22 out of 100 heat-repressible RNA-based thermosensor in toolkit.
·Activities of thermosensors decrease at elevated temperature
We measured the activities of these thermosensors at 3 temperatures: 29, 37 and 42℃. Figure 1 shows the measurement results of the 22 different thermosensors. Compared with the positive control, all the heat-repressible RNA-based thermosensors' normalized fluorescence decrease at elevated temperature. They also have different intensity and sensitivity. We have added the characterized result to the parts registry, users can choose their appropriate thermosensors.
Figure 1. Experimental measurement of the heat-repressible RNA-based thermosensors show a variety of responses. (A) Rows represent activity levels of different thermosensors. These values are normalized using the fluorescence/Abs600 of positive control. (B) Replotting of data from (A). Each set of three bars represents the activity level of a different thermosensor. The bar colors purple, yellow and red represent the temperature 29, 37 and 42℃.
We also computed the fold-change from 29 to 37℃ and 37 to 42℃. As the figure shows, these fold-changes were lower than positive control and the fluorescence per Abs600 were higher than negative control-2.
Figure 2. Fold-change of the heat-inducible RNA-based thermosensors. (A, B) Each blue dot represents an individual thermosensor. The red horizontal represents the fold-change of positive control. The red vertical line represents the normalized fluorescence of negative control.
Conclusion: As these experiment results show, the heat-repressible RNA-based thermosensors work really well. The activities decrease with temperature increases. Additionally, the difference in fluorescence intensity and the rate of decrease points to the diversity in thermosensor response. We consider the sequence change in stem length, loop size, and mismatches or bulges in the stem cause the differences in thermosensor response. These are the results of SynRT toolkit 2.0.>Click here to see the results of version 3.0<