Revision as of 13:52, 13 October 2018

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 low temperatures.

Here, we designed short, heat-repressible RNA-based thermosensors. These thermosensor sequences contain a single-strand RNase E cleavage (RC) site. RNase E is 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.

Figure 1. Mechanism of heat-repressible RNA-based thermosensors.

2. Design

In order to design heat-repressible RNA-based thermosensores with different melting temperatures, intensity and sensitivity, we change the ARC sequence because the RC sequence is conserved. Three structural parameters come into consideration: stem length, loop size and mismatches or bulges in the stem.

Stem length is determined by ARC sequence. Adding stem length can optimize heat-repressible RNA-based thermosensors to higher temperature, while decreasing stem length has the opposite effect. The stem length is 13 base paring in K2541101. Loop size can moderate thermosensors melting temperature to a appropriate temperature. In K2541101, the loop sequence is AAUAA. Furthermore, we change base composition in ARC sequence to decrease melting temperature. We make a mismatch in the fifth base by changing it from G to U. And we make a bulge in the ninth base by inserting U. After designing, the theromsensor sequence is predicted by computational methods mFOLD to get its minimum free energy and secondary structure.

Figure 2. Design of synthetic heat-repressible RNA-based thermosensor. (A) The RNA secondary structure is predictred by mFOLD. (B) 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 double terminator (BBa_B0010 and BBa_B0012). We select a constitutive Anderson promoter J23106 as a appropriate promoter by pre-experiment. The sfGFP_optimism has faster folding speed and higher fluorescence intensity. The double terminator can reduce leakge (Figure 3). We characterized RNA-based thermosensors in E.coli DH5a.

Figure 3. The measurement device.

K2541101, K2415109, K2541119 and K2541101 are four different heat-repressible RNA-based thermosensors. pos.control is positive control. The final normalized fluorescence was calculated as follows: normalized fluorescence = [(Fluorescence/Abs₆₀₀)_TS - (Fluorescence/Abs₆₀₀)_neg] / [(Fluorescence/Abs₆₀₀)_pos - (Fluorescence/Abs₆₀₀)_neg] ( TS = thermosensor, pos = positive control, and neg = BBa_J364007 ). As shown in figure 4, the fluorescence intensity of K2541101 decreases with elevated temperature.

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.

Figure 5. 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 SynRT toolkit for the regulation of gene expression.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 31

@@ Line 11: / Line 11: @@
 <h5>
 <P style="text-indent:2em;">
-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.
+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 low temperatures.
 </p>
 <P style="text-indent:2em;">
-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'.
+Here, we designed short, heat-repressible RNA-based thermosensors. These thermosensor sequences contain a single-strand RNase E cleavage (RC) site. RNase E is 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’.
 </p>
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@@ Line 255: / Line 255: @@
 <h5>
 <P style="text-indent:2em;">
-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.
+In order to design heat-repressible RNA-based thermosensores with different melting temperatures, intensity and sensitivity, we change the ARC sequence because the RC sequence is conserved. Three structural parameters come into consideration: stem length, loop size and mismatches or bulges in the stem.
 </p>
 <P style="text-indent:2em;">
-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.
+Stem length is determined by ARC sequence. Adding stem length can optimize heat-repressible RNA-based thermosensors to higher temperature, while decreasing stem length has the opposite effect. The stem length is 13 base paring in K2541101. Loop size can moderate thermosensors melting temperature to a appropriate temperature. In K2541101, the loop sequence is AAUAA. Furthermore, we change base composition in ARC sequence to decrease melting temperature. We make a mismatch in the fifth base by changing it from G to U. And we make a bulge in the ninth base by inserting U. After designing, the theromsensor sequence is predicted by computational methods mFOLD to get its minimum free energy and secondary structure.
-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.) After designing, the theromsensor sequence is predicted by computational methods mFOLD to get its minimum free energy and secondary structure (figure 2).
 </p>
 </h5>
 [[File:K2541101 f2.png|center|K2541101 f2]]
-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, ΔG and Tm are in the table.
+Figure 2. Design of synthetic heat-repressible RNA-based thermosensor. (A) The RNA secondary structure is predictred by mFOLD. (B) ARC sequence, loop sequence, the site of mismatch or bulge in the stem, △G and Tm are in the table.
 <h1>'''3.Characterization'''</h1>
 <h5>
 <P style="text-indent:2em;">
-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 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.
+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 double terminator (BBa_B0010 and BBa_B0012). We select a constitutive Anderson promoter J23106 as a appropriate promoter by pre-experiment. The sfGFP_optimism has faster folding speed and higher fluorescence intensity. The double terminator can reduce leakge (Figure 3). We characterized RNA-based thermosensors in ''E.coli'' DH5a.
 </p>
 </h5>
@@ Line 282: / Line 280: @@
 <h5>
 <P style="text-indent:2em;">
-On the left side of the figure 4 is K2541101. On the right of figure 4 is positive control. K2415109, K2514119 and K2514101 are three heat-repressible RNA-based thermosensors with different intensity. As shown in the figure 4, the thermosensor K2541001 is ‘off’ from 29°C to 37°C. Its intensity is higher than K2514119.
+K2541101, K2415109, K2541119 and K2541101 are four different heat-repressible RNA-based thermosensors. pos.control is positive control. The final normalized fluorescence was calculated as follows: normalized fluorescence = [(Fluorescence/Abs<sub>600</sub>)<sub>TS</sub> - (Fluorescence/Abs<sub>600</sub>)<sub>neg</sub>] / [(Fluorescence/Abs<sub>600</sub>)<sub>pos</sub> - (Fluorescence/Abs<sub>600</sub>)<sub>neg</sub>] ( TS = thermosensor, pos = positive control, and neg = BBa_J364007 ). As shown in figure 4, the fluorescence intensity of K2541101 decreases with elevated temperature.
 </p>
 </h5>
@@ Line 292: / Line 290: @@
 [[File:RE figure6.png|center|RE figure6]]
 Figure 5. 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.
 <h1>'''4. Conclusion'''</h1>
 <h5>
 <P style="text-indent:2em;">
-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.
+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 SynRT toolkit for the regulation of gene expression.
 </p>
 </h5>