Difference between revisions of "Part:BBa K3247000"
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===Design Notes===
 
===Design Notes===
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The stem-loop structure of the thermometer was created by taking the complement of the ribosome binding site (RBS) and adding nucleotides to either side. The additional bases were mutated; the resulting altered sequence is known as the variable region. Mutating the variable region keeps the RBS intact while modifying the RNA thermometer secondary structure to change the melting temperature. There is no internal BsaI cut site, since it would render the thermometer incompatible with our assembly method.  
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The stem-loop structure of the thermometer was created by taking the complement of the ribosome binding site (RBS) and adding nucleotides to either side. The additional bases were mutated; the resulting altered sequence is known as the variable region. Mutating the variable region keeps the RBS intact while modifying the RNA thermometer secondary structure to change the melting temperature. There is no internal BsaI cut site, since it would render the thermometer incompatible with our assembly method.
  
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<img src="https://2019.igem.org/wiki/images/0/07/T--Rice--0125.png" width = 60%>
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<p><b>Figure 1a.</b>Predicted RNA thermometer structure at 25 °C.</p>
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<img src="https://2019.igem.org/wiki/images/5/50/T--Rice--0130.png" width = 60%>
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<p><b>Figure 1b.</b>Predicted RNA thermometer structure at 30 °C.</p>
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<img src="https://2019.igem.org/wiki/images/0/06/T--Rice--0137.png" width = 60%>
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<p><b>Figure 1c.</b>Predicted RNA thermometer structure at 37 °C.</p>
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===Characterization===
 
===Characterization===
 
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<img src="https://2019.igem.org/wiki/images/3/39/T--Rice--Nochill1.png" width = 60%>
 
<img src="https://2019.igem.org/wiki/images/3/39/T--Rice--Nochill1.png" width = 60%>
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<p><b>Figure 1.</b> Fluorescence intensity at 25 °C, 30 °C, and 37 °C.</p>
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<p><b>Figure 2.</b> Fluorescence intensity at 25 °C, 30 °C, and 37 °C.</p>
 
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Revision as of 23:15, 21 October 2019

RNA Thermometer NoChill-01


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 7
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 7
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 7
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The stem-loop structure of the thermometer was created by taking the complement of the ribosome binding site (RBS) and adding nucleotides to either side. The additional bases were mutated; the resulting altered sequence is known as the variable region. Mutating the variable region keeps the RBS intact while modifying the RNA thermometer secondary structure to change the melting temperature. There is no internal BsaI cut site, since it would render the thermometer incompatible with our assembly method.

Figure 1a.Predicted RNA thermometer structure at 25 °C.

Figure 1b.Predicted RNA thermometer structure at 30 °C.

Figure 1c.Predicted RNA thermometer structure at 37 °C.

Characterization

Figure 2. Fluorescence intensity at 25 °C, 30 °C, and 37 °C.

Source

The thermometers were designed de novo using NUPACK and VSAlgorithm


References