Difference between revisions of "Part:BBa K2541006"
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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-inducible RNA-based thermosensors can initiate translation of downstream genes at high temperatures.
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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.
 
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Heat-inducible RNA-based thermosensors are RNA genetic control systems that sense temperature changes. At low temperatures, the mRNA adopts a stem-loop conformation that masks the Shine–Dalgarno (SD) sequence within the 5’-untranslated region (5’-UTR) and, in this way, prevents ribosome binding and translation. At elevated temperatures, the RNA secondary structure melts locally, thereby giving the ribosomes access to the SD sequence to initiate translation (Figure 1). Whereas natural RNA-based thermosensors have a relatively complicated secondary structure with multiple stems, hairpin loops and bulges which impeds application process.
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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.  
 
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Our team designed synthetic heat-inducible RNA-based thermosensors that are considerably simpler than naturally occurring thermosensors and can be exploited as convenient on/off switches of gene expression.
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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’.
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These short, modular heat-repressible RNA-based thermosensors can be exploited as convenient on/off switches of gene expression.
 
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       <text class="d" transform="translate(349.01 185.45)">SD Sequence</text>
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     <text id="ribosome_text" class="h" transform="translate(385.48 175.45)">RNAse E</text>
  
 
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    <p>Figure 1. Mechanism of heat-repressible RNA-based thermosensors.</p>
 
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  <p>Figure 1. Mechanism of heat-inducible RNA-based thermosensors.</p>
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The heat-inducible RNA-based theromsensors are designed on the basis of the melting temperature of the minimum free energy structure, consisting of ASD (anti-SD) sequence, loop sequence and consensus SD sequence (5’-AAGGAG-3’). To optimize the thermosensors for the desired melting temperature, intensity and sensitivity, three structural parameters come into consideration: stem length, loop size and mismatches or bulges in the stem.
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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.
 
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Stem length is determined by ASD sequence because the SD sequence is conserved. Adding stem length can optimize heat-inducible RNA-based thermosensors to more high temperature, while decreasing stem length has the opposite effect. The stem length is 7 base parings in K2541006. Loop size can moderate thermosensors melting temperature to a suitable temperature. In K2541006, the loop sequence is AAUAA. Finally, we get the sequence as the figure 2B. After designing, the theromsensor sequence is predicted by computational methods mFOLD to get its minimum free energy and secondary structure (figure 2).
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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 parings in K2541106. Loop size can moderate thermosensors melting temperature to a appropriate temperature. In K2541106, the loop sequence is AAAAAUAUAUAUAAAA. 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.
 
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[[File:K2541006 f2.png|center|K2541006 f2]]
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[[File:K2541106 f2.png|center|K2541106 f2]]
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Figure 2. Design of synthetic heat-inducible RNA-based thermosensor. (A) The RNA secondary structure is predictred by mFOLD. (B) Thermosensor sequence, ASD sequence, loop sequence, the site of mismatch or bulge in the stem and ΔG are in the table.
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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, ΔG and Tm are in the table.
  
 
<h1>'''3.Characterization'''</h1>
 
<h1>'''3.Characterization'''</h1>
 
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The thermosensor sequence is constructed on the pSB1C3 vector by GoldenGate assembly. The measurement device is composed of Anderson promotor (BBa_J23104), thermosensor (BBa_K2541006), sfGFP_optimism (BBa_K2541400) and double terminator (BBa_B0010 and BBa_B0012). We select a constitutive Anderson promoter J23104 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.
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The thermosensor sequence is constructed on the pSB1C3 vector by GoldenGate assembly. The measurement device is composed of Anderson promotor (BBa_J23106), thermosensor (BBa_K2541106), 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.
 
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K2541006, K2415029, K2541013 and K2541037 are four different heat-inducible 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 K2541006 increases with elevated temperature.
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K2541106, 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 K2541106 decreases with elevated temperature.
 
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[[File:K2541006 f4.png|center|K2541006 f4]]
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[[File:K2541106 f4.png|center|K2541106 f4]]
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Figure 4. Characteristics of synthetic heat-inducible RNA-based thermosensors. Each set of six bars represents the activity level of a different thermosensor. The bar colors purple, aquamarine, light green, orange, red and brown represent the temperatures 29, 31, 35, 37, 39 and 42°C, respectively. The height of the bars corresponds to the normalized fluorescence.
+
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.
 
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[[File:RE figure6.png|center|RE figure6]]
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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.
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This year, our project 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. We studied a set of measured synthetic RNA-based thermosensors, finding consistency among our experimental and computational analyses.
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[[File:K2514006 f5.png|center|K2514006 f5]]
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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 melting temperature, intensity, threshold and sensitivity are emphasized.
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[[File:RT figure6.png|center|caption]]
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Figure 6. Experimental measurements of the collection of heat-inducible RNA-based thermosensors show a variety of responses. (A) Rows represent activity levels of different thermosensors. (B) Each set of six bars represents the activity level of a different thermosensor. The bar colors purple, aquamarine, light green, orange, red and brown represent the temperatures 29, 31, 35, 37, 39 and 42°C, respectively. The height of the bars corresponds to the normalized fluorescence.
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<h1>'''4. Conclusion'''</h1>
 
<h1>'''4. Conclusion'''</h1>
 
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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 masking the Shine–Dalgarno (SD) sequence, thus providing useful SynRT toolkit for the regulation of gene expression.
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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.
 
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Revision as of 03:28, 14 October 2018


Heat-inducible RNA-based thermosensor-6

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 exploited as convenient on/off switches of gene expression.

RC anti-RC RNAse E

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 parings in K2541106. Loop size can moderate thermosensors melting temperature to a appropriate temperature. In K2541106, the loop sequence is AAAAAUAUAUAUAAAA. 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.

K2541106 f2

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, ΔG 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_K2541106), 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.

caption

Figure 3. The measurement device.


K2541106, 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 K2541106 decreases with elevated temperature.

K2541106 f4

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.


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.

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
    COMPATIBLE WITH RFC[1000]