Difference between revisions of "Part:BBa K2541106"
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2541106 short</partinfo>
 
<partinfo>BBa_K2541106 short</partinfo>
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<h5>
 +
<P style="text-indent:2em;">
 +
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.
 +
</p>
 +
</h5>
  
−
A RNA 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 thermosensors can repress translation of downstream genes at high temperatures.
+
<h1>'''1. Usage and Biology'''</h1>
 +
<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 low temperatures.
 +
</p>
 +
<P style="text-indent:2em;">
 +
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>
 +
<P style="text-indent:2em;">
 +
These short, modular heat-repressible RNA-based thermosensors can be exploited as convenient on/off switches of gene expression.
 +
</p>
 +
</h5>
 +
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 +
  <div class="stem-loop hot-repressive">
  
−
<h1>'''Usage and Biology'''</h1>
+
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−
RNA-based temperature sensing is common in bacteria that live in fluctuating environments. Most naturally-occurring RNA thermosensors are heat-inducible, have long sequences, and function by sequestering the ribosome binding site in a stem-loop structure at lower temperatures. Here, we designed short, heat-repressible RNA thermosensors. These thermosensors contain a single-strand RNA cleavage site for RNase E, an enzyme native to Escherichia coli and many other organisms, in the 5' untranslated region of the target gene. At low temperatures, the cleavage site is sequestered in a stem-loop, and gene expression is unobstructed. At elevated temperatures, the stem-loop unfolds, allowing for mRNA degradation and turning off expression. These short, modular heat-repressible RNA thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications.
+
  
−
<!-- -->
 
−
<h1>'''Characterization'''</h1>
 
−
The thermosensor is constructed on the pSB1C3 vector by GoldenGate assembly. As shown below, the measurement device is composed of Anderson promotor (BBa_J23104), thermosensor (BBa_K2541106) and sfGFP(BBa_K2541400). We measured the sfGFP expression to get the state of the heat-repressible RNA thermosensor at different temperatures.
 
  
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As shown in the figure, the thermosensor is "off" at [   ]. Our data show that efficient RNA thermosensors can be built from a single small RNA stem-loop structure masking the ribosome binding site, thus providing useful RNA-based toolkit for the regulation of gene expression.
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    <p>Figure 1. Mechanism of heat-repressible RNA-based thermosensors.</p>
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 +
 
 +
<h1>'''2. Design'''</h1>
 +
<h5>
 +
<P style="text-indent:2em;">
 +
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. 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.
 +
</p>
 +
</h5>
 +
[[File:K2541106 f2.png|center|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.
 +
 
 +
<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_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.
 +
</p>
 +
</h5>
 +
[[File:measurement device figure3.png|center|caption]]
 +
<html><center>
 +
Figure 3. The measurement device.
 +
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 +
</html>
 +
----
 +
 
 +
 
 +
<h5>
 +
<P style="text-indent:2em;">
 +
K2541105, 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 K2541105 decreases with elevated temperature.
 +
</p>
 +
</h5>
 +
[[File:K2541106 f4.png|center|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.
 +
----
 +
 
 +
 
 +
[[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 SynRT toolkit for the regulation of gene expression.
 +
</p>
 +
</h5>
  
  

Revision as of 03:38, 14 October 2018


Heat-repressible 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.


K2541105, 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 K2541105 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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 42