Difference between revisions of "Team:Jilin China/Result/Version 3"

Results

We added 10 cold-reprssible RNA-based thermosensors and 8 cold-inducible RNA-based thermosensors to the SynRT Toolkit. After that, we named the updated toolkit as SynRT Toolkit version 3.0.

Cold-repressible RNA-based thermosensors

Cold-repressible RNA-based thermosensors are designed based on the RNase III. We constructed their measurement devices and measured the activities at five temperatures: 15, 25, 29, 35 and 37℃. The measured temperature range is lower than the heat-inducible RNA-based thermosensors' temperature range. We also designed a new negtive control, which always has a cleavage site of RNase III, so it will be digest by the enzyme. After measurement, we chose 10 out of 50 thermosensors.

Figure 1 shows the change of activities of different thermosensors. We find that all of these thermosensors' activities decrease with temperature decreasing.

Figure 1. Expermimental measurement of the cold-repressible RNA-based thermosensors show a variety of responses. (A) Rows represent activity levels of different thermosensors. The activity levels are the mean of three replications. These values are normalized using the fluorescence/Abs600 of pos.control. (B) Replotting of data from (A). Each set of five bars represents the activity level of a different thermosensor. The bar colors purple, green, yellow, orange and red represent the temperature 15, 25, 29, 35 and 37℃. The height of the error bars corresponds to the mean levels of three replications with the standard deviation.

For most of the heat-inducible RNA-based thermosensors, the sensing temperature range is between 30 to 40. However, for the cold-repressible RNA-based thermosensors, the sensing temperature range decrease to 15~35℃.

Figure 2. Fold-change of the cold-repressible 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.

Discussion: As these results show, cold-repressible RNA-based thermosensors work really well. Their activities decrease with the temperature decreasing. And the sensing temperature range is different from heat-inducible RNA-based thermosensors'. Cold repressible RNA-based thermosensor can sense lower temperature.

Cold-inducible RNA-based thermosensors

Cold-inducible RNA-based thermosensors are designed based on the cspA 5'UTR mRNA, by changing psedoknot length, GC content, Wobble base pair, we designed 50 different thermosensors, and measured their activity in 15, 20 and 25℃. According to the result, we screened 8 out of 50 thermosensors.

As the figure 3 shows, all the thermosensors' activities decrease at elevated temperature.

Figure 3. Expermimental measurement of the cold-inducible RNA-based thermosensors show a variety of responses. (A) Rows represent activity levels of different thermosensors. The activity levels are the mean of three replications. These values are normalized using the fluorescence/Abs600 of pos.control. (B) Replotting of data from (A). Each set of five bars represents the activity level of a different thermosensor. The bar colors purple, green, yellow, orange and red represent the temperature 15, 20 and 25℃. The height of the error bars corresponds to the mean levels of three replications with the standard deviation.

The sensing temperature range of cold-inducible RNA-based thermosensors is lower than heat-repressive RNA-based thermosensors. As the figure 4 shows, most of the cold-inducible RNA-based thermosensors' activity decrease sharply from 15 to 20℃. After that, the change of activity gradually levels off.

Figure 4. Fold-change of the cold-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.

As these results show, cold-induced RNA-based thermosensors can work. Their activities decrease at elevated temperature. And the sensing temperature range is lower than heat-repressible RNA-based thermosensors'.

To sum up: Our SynRT toolkit version 3.0, including heat-inducible RNA thermosensors, heat-repressible RNA-based thermosensors, cold-inducible RNA-based thermosensors and cold-repressible RNA-based thermosensors can work under realistic conditions. You can read more in our Demonstrate page.