Difference between revisions of "Part:BBa K2541407"

 
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<partinfo>BBa_K2541407 short</partinfo>
 
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A RNA thermosensor that can be used for temperature sensitive post-transcriptional regulation which is based on the change of RNA sencondary structure. The cold-repressible RNA thermosensors can repress translation of downstream genes at low temperatures. The composite part is composed of promoter BBa_J23106, cold-repressible RNA thermosensor-4 BBa_K2541204, reporetr protein sfGFP BBa_K2541400 and terminator BBa_B0015.
  
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<h1>'''Usage and Biology'''</h1>
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===Usage and Biology===
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RNA-based temperature sensing is common in bacteria that live in fluctuating environments. Most naturally-occurring RNA thermosensors have long sequences and complicated sencondary structure. Here, we designed short, cold-repressible RNA thermosensors, which will form a stem-loop upstream Shine–Dalgarno (SD) sequence. These thermosensors contain a double-strand RNA cleavage site for RNase III, an enzyme native to Escherichia coli and many other organisms, in the 5' untranslated region of the target gene. At low temperatures, the mRNA stem-loop is stable to expose the RNase III cleavage site and the transcript will be degraded. At elevated temperatures, the stem-loop will unfold and translation will occur unhindered. These short, modular cold-repressible RNA thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications.
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The composite part can be used as a measurement device for different cold-repressible RNA thermosensors. Wu use goldengate assembly to change differrnt thermosensors to measure their melting temperature. sfGFP (BBa_K2541400) is BbsI restriction site free, so it can be used in goldengate assembly to achieve efficient and rapid assembly of gene fragments.
  
 
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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_K2541204) and  sfGFP (BBa_K2541400) and terminator (BBa_B0015). We measured the sfGFP expression to get the state of the cold-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>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2541407 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2541407 SequenceAndFeatures</partinfo>

Revision as of 05:22, 10 October 2018


Cold-repressible RNA thermosensor measurement device

A RNA thermosensor that can be used for temperature sensitive post-transcriptional regulation which is based on the change of RNA sencondary structure. The cold-repressible RNA thermosensors can repress translation of downstream genes at low temperatures. The composite part is composed of promoter BBa_J23106, cold-repressible RNA thermosensor-4 BBa_K2541204, reporetr protein sfGFP BBa_K2541400 and terminator BBa_B0015.

Usage and Biology

RNA-based temperature sensing is common in bacteria that live in fluctuating environments. Most naturally-occurring RNA thermosensors have long sequences and complicated sencondary structure. Here, we designed short, cold-repressible RNA thermosensors, which will form a stem-loop upstream Shine–Dalgarno (SD) sequence. These thermosensors contain a double-strand RNA cleavage site for RNase III, an enzyme native to Escherichia coli and many other organisms, in the 5' untranslated region of the target gene. At low temperatures, the mRNA stem-loop is stable to expose the RNase III cleavage site and the transcript will be degraded. At elevated temperatures, the stem-loop will unfold and translation will occur unhindered. These short, modular cold-repressible RNA thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications.

The composite part can be used as a measurement device for different cold-repressible RNA thermosensors. Wu use goldengate assembly to change differrnt thermosensors to measure their melting temperature. sfGFP (BBa_K2541400) is BbsI restriction site free, so it can be used in goldengate assembly to achieve efficient and rapid assembly of gene fragments.

Characterization

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_K2541204) and sfGFP (BBa_K2541400) and terminator (BBa_B0015). We measured the sfGFP expression to get the state of the cold-repressible RNA thermosensor at different temperatures.


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.


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
    COMPATIBLE WITH RFC[1000]