Difference between revisions of "Part:BBa K2541406"
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2541406 short</partinfo>
 
<partinfo>BBa_K2541406 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. The composite part is a measurement device, consisting of Anderson promotor (BBa_J23106), heat-repressible RNA-based  thermosensor-1 (BBa_K2541101), sfGFP_optimism (BBa_K2541400) and double terminator (BBa_B0010 and BBa_B0012).</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. The composite part is composed of promoter BBa_J23106, heat-repressible RNA thermosensor-1 BBa_K25411101, reporetr protein sfGFP BBa_K2541400 and terminator BBa_B0015.
+
<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’. These short, modular heat-repressible RNA-based thermosensors can be exploited as convenient on/off switches of gene expression.
 +
</p>
 +
<P style="text-indent:2em;">
 +
Green fluorescent protein (GFP) is commonly used as a reporter gene in intact cells and organisms. This year we select sfGFP (BBa_K2541400), a robustly folded version of GFP, called superfolder GFP as a reporter protein. Compared to superfolder GFP (BBa_I746916), sfGFP_optimism (BBa_K2541400) is BbsI restriction site free, so it can be used in GoldenGate assembly to achieve efficient and rapid assembly of gene fragments. And sfGFP_optimism (BBa_K2541400) has stronger fluorescence intensity than superfolder GFP (BBa_I746916).
 +
</p>
 +
</h5>
 +
<html><center>
 +
  <div class="stem-loop hot-repressive">
  
−
<h1>'''Usage and Biology'''</h1>
+
    <input id="checked_2" type="checkbox" class="switch" />
−
== Heat-repressible RNA thermosensor-1 ==
+
−
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.
+
  
−
== sfGFP ==
 
−
Green fluorescent protein (GFP) exhibits intrinsic fluorescence and is commonly used as a reporter gene in intact cells and organisms. Many mutants of the protein with either modified spectral properties, increased fluorescence intensity, or improved folding properties have been reported.
 
  
−
GFP often misfold when expressed as fusions with other proteins, while a robustly folded version of GFP, called superfolder GFP, was developed and described by Pédelacq et al at 2006 that folds well even when fused to poorly folded polypeptides. There is another superfolder GFP designed by Overkamp W et al at 2013, which is codon optimized for S. pneumoniae. It was be used in Escherichia coli by Segall-Shapiro T H et al at 2018.
+
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−
This year our team registered the superfolder GFP designed by Overkamp W et al with a BBa_K2541400 (called sfGFP). Compared to superfolder GFP(BBa_I746916), sfGFP (BBa_K2541400) is BbsI restriction site free, so it can be used in GoldenGate assembly to achieve efficient and
+
.switch::before{
−
rapid assembly of gene fragments. And sfGFP (BBa_K2541400) has stronger fluorescence intensity than superfolder GFP(BBa_I746916).
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−
== Conclusion ==
+
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−
The composite part can be used as a measurement device for different heat-repressible RNA thermosensors. Wu use GoldenGate assembly to change differrnt thermosensors to measure their melting temperature.
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−
<h1>'''Characterization'''</h1>
+
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−
The thermosensor is constructed on the pSB1C3 vector by GoldenGate assembly. As shown below, the measurement device is composed of Anderson promotor (BBa_J23106), thermosensor (BBa_K2541101) and sfGFP (BBa_K2541400) and terminator (BBa_B0015). 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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<span class='h3bb'>Sequence and Features</span>
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 +
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 +
    <p>Figure 1. Mechanism of heat-repressible RNA-based thermosensors.</p>
 +
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 +
 +
 +
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 +
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 +
</html>
 +
 +
<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 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.
 +
</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) 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_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>
 +
[[File:measurement device figure3.png|center|caption]]
 +
<html><center>
 +
Figure 3. The measurement device.
 +
  </script></center>
 +
</html>
 +
----
 +
 +
 +
<h5>
 +
<P style="text-indent:2em;">
 +
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>
 +
[[File:K2541101 f4 new.png|center|K2541101 f4 new]]
 +
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>
 +
 +
 +
<span class='h3bb'>Sequence and Features</span>
 +
<partinfo>BBa_K2541406 SequenceAndFeatures</partinfo>
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2541406 parameters</partinfo>
 
<partinfo>BBa_K2541406 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Revision as of 07:09, 14 October 2018


Heat-repressible RNA-based thermosensor measurement device

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. The composite part is a measurement device, consisting of Anderson promotor (BBa_J23106), heat-repressible RNA-based thermosensor-1 (BBa_K2541101), sfGFP_optimism (BBa_K2541400) and double terminator (BBa_B0010 and BBa_B0012).

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.

Green fluorescent protein (GFP) is commonly used as a reporter gene in intact cells and organisms. This year we select sfGFP (BBa_K2541400), a robustly folded version of GFP, called superfolder GFP as a reporter protein. Compared to superfolder GFP (BBa_I746916), sfGFP_optimism (BBa_K2541400) is BbsI restriction site free, so it can be used in GoldenGate assembly to achieve efficient and rapid assembly of gene fragments. And sfGFP_optimism (BBa_K2541400) has stronger fluorescence intensity than superfolder GFP (BBa_I746916).

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 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.

K2541101 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_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.

caption

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.

K2541101 f4 new

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
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 539
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 74