Part:BBa_K2541101
Heat-repressible RNA-based thermosensor-1
A RNA-based thermosensor that can be used for temperature sensitive post-transcriptional regulation which is based on the change of RNA sencondary structure. The heat-repressible RNA-based thermosensors can repress translation of downstream genes at high temperatures.
1. Usage and Biology
RNA-based temperature sensing is common in bacteria that live in fluctuating environments. Most naturally occurring RNA-based thermosensors are heat inducible, have long sequences, and function by sequestering the Shine–Dalgarno (SD) sequence in a stem-loop structure at lower temperatures.
Here, we designed short, heat-repressible RNA-based thermosensors. These thermosensors contain a single-strand RNA cleavage (RC) site for RNase E, an enzyme native to Escherichia coli and many other organisms. Each heat-repressible RNA-based thermosensor sequence was inserted downstream of the transcription start site and upstream of the SD sequence. At high temperatures, the RNase E cleavage site (RC) is exposed, mRNA was cleaved by RNase E, and expression is ‘OFF’. At low temperatures, the RC binds to the anti-RNase E cleavage site (ARC) and forms a stem-loop. This structure sequesters the RC, and expression is ‘ON’.
These short, modular heat-repressible RNA-based thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications.
2. Design
The RC and ARC were considered to be the two separate strands. We keep the RC sequence conserved whie changing the ARC sequence. In order to obtain heat repressible RNA-based thermosensores with different melting temperatures, intensity and sensitivity, we change the stem length, loop size and the mismatches or bulges in the stem.
Stem length is determined by ARC sequence because the RC sequence is conserved. Adding stem length can optimize heat repressible RNA-based thermosensors to more high temperature, while decreasing stem length has the opposite effect. The stem length is X base paring in K2541101.
Loop size can moderate thermosensors melting temperature to a suitable temperature. In K2541101, the loop sequence is (选一个) (AAUAA,AAAUAA, AAAAUAUAAA,AAAAAAUAUAAA, AAAAAUAUAUAUAAAA).
Furthermore, we change base composition in ARC sequence to decrease response temperature. We make a mismatch in the sixth base by changing it from A to T. (or We make a bulge in the sixth base by deleting or inserting A.)
Finally, we get the sequence as the figure 2.
Figure 2. Design of synthetic heat repressible RNA-based thermosensor. (A) The RNA secondary structure is predictred by mFOLD. (B) Thermosensor sequence, ARC sequence, loop sequence, the site of mismatch or bulge in the stem, △G and Tm are in the table.
3.Characterization
The thermosensor sequence is constructed on the pSB1C3 vector by GoldenGate assembly. The measurement device is composed of Anderson promotor (BBa_J23106), thermosensor (BBa_K2541101), sfGFP_optimism (BBa_K2541400) and terminator (BBa_B0015). We select a medium constitutive Anderson promoter J23106 as a suitable promoter by doing pre-experiment. The sfGFP_optimism has faster folding speed and higher fluorescence intensity. The B0015 is a double terminator that can reduce leakge (Figure 3). We characterized RNA-based thermosensors in E.coli DH5a.
On the left side of the figure 4 is K2541101. On the right of figure 4 is positive control. K2415029, K2514013, K2514037 are three heat inducible RNA-based thermosensors with different intensity. The final normalized fluorescence was calculated as follows: normalized fluorescence = [(GFP/Abs)TS - (GFP/Abs)neg] / [(GFP/Abs)pos - (GFP/Abs)neg] ( TS = thermosensor, pos = positive control, and neg = E.coli DH5a ). As shown in the figure 4, the thermosensor K2541001 melting temperature range is ‘off’ from 29°C to 37°C. Its intensity is about equal to K2541037.
Figure 4. Characteristics of synthetic heat repressible RNA-based thermosensors. Each set of three bars represents the activity level of a different thermosensor. The bar colors purple, yellow and red represent the temperatures 29, 37 and 42°C, respectively. The height of the bars corresponds to the normalized fluorescence.
This year, our objective is to design a collection of RNA-based thermosensors with different melting temperatures, intensity and sensitivity. We used a combination of experimental measurements and computations of RNA secondary structures to achieve this objective. We studied a set of measured synthetic RNA-based thermosensors, finding consistency with measured results and among our experimental and computational analyses.
Figure 5. Temperature response of the heat inducible RNA-based thermosensor. The red solid line represents the activity of the RNA-based thermosensor as a function of temperature. Key quantitative features of the response such as Tm, intensity, threshold and sensitivity are emphasized.
Figure 6. Experimental measurements of the collection of heat repressible RNA-based thermosensors show a variety of responses. (A) Rows represent activity levels of different thermosensors. (B) Each set of three bars represents the activity level of a different thermosensor. The bar colors purple, yellow and red represent the temperatures 29, 37 and 42°C, respectively. The height of the bars corresponds to the normalized fluorescence.
4. Conclusion
Our data show that efficient RNA-based thermosensors with different melting temperatures, intensity and sensitivity can be built from a single small RNA stem-loop structure, thus providing useful RNA-based toolkit for the regulation of gene expression.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
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- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 31
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