Difference between revisions of "Part:BBa K115016:Design"

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===Design Notes===
 
===Design Notes===
  
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The designed sequence is based on a ROSE RNA thermometer retrieved from ''Bradyrhizobium japonicum USDA 110'' . The temperature sensitive hairpin of this RNA thermometer is destabilized in order to get a lower switching temperature. Incorporation of weak base pairs, bulge loops, and internal loops is used for the destabilization, while ensuring that the secondary structure remains unaltered, and the distribution of the free energy within the temperature sensitive hairpin remains similar to the characteristic distribution which seems to be similar to all RNA thermometers of this type as shown [http://2008.igem.org/Team:TUDelft/Temperature_analysis#Results here]. The dark blue line in the graph shows the found trend which is the characteristic distribution of free energy within 32 RNA thermometers which have their switching point around 37°C. The light blue shows the distribution of the free energy for this part at 27°C (as calculated with RNAeval). This line is fitted to the trend line, so that its temperature sensitive hairpin has the same stability, but at a lower temperature (27°C instead of 37°C). The assumption is that this will also cause the switching temperature to 27°C.
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The designed sequence is based on a [http://en.wikipedia.org/wiki/Repression_of_heat_shock_gene_expression_(ROSE)_element ROSE] RNA thermometer retrieved from ''Bradyrhizobium japonicum USDA 110'' . The temperature sensitive hairpin of this RNA thermometer (the one that contains the Shine Dalgarno (SD) sequence) is destabilized in order to get a lower switching temperature.  
  
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The secondary structure of the part after ligation to a protein coding part, as predicted by RNAfold, is given at the right of the figure. The light blue region is the Shine Dalgarno region, which is the ribosome binding site.
+
Incorporation of weak base pairs, bulge loops, and internal loops is used for the destabilization of the temperature sensitive hairpin, while ensuring that the secondary structure remains unaltered, and the distribution of the free energy within the temperature sensitive hairpin remains similar to the [http://2008.igem.org/Team:TUDelft/Temperature_analysis#Results characteristic free energy distribution] as found for 32 ROSE RNA thermometers. The dark blue line in the graph shows the found characteristic free energy distribution of the 32 ROSE RNA thermometers which have their switching point around 37°C. The light blue line shows the distribution of the free energy for this part at 27°C. This line is fitted to the trend line, so that the temperature sensitive hairpin of this part has the same stability, but at a lower temperature (27°C instead of 37°C). The assumption is that this will also cause the switching temperature to drop to 27°C.
 +
 
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The secondary structure of the part after ligation to a protein coding part, as predicted by [http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi RNAfold], is shown at the left. Notice that the 3' end, including the scar and the start codon, does not belong to the part. The blue region is the Shine Dalgarno sequence which is the ribosome binding site.
  
 
See our [http://2008.igem.org/Team:TUDelft/Temperature_design2 wiki] for an extended description of the design of this part.
 
See our [http://2008.igem.org/Team:TUDelft/Temperature_design2 wiki] for an extended description of the design of this part.
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===Source===
 
===Source===
  
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''Bradyrhizobium japonicum USDA 110'' ([http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genome&Cmd=ShowDetailView&TermToSearch=272 <code>NC_004463</code>]), residing at location [http://www.ncbi.nlm.nih.gov/projects/sviewer/?id=NC_004463.1&v=5784144..5784239 <code>5784144-5784239</code>] within the genome, which is at the 5' side of the gene [http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=search&term=NC_004463%20hspb hspB].
+
RNA thermometer from ''Bradyrhizobium japonicum USDA 110'' ([http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genome&Cmd=ShowDetailView&TermToSearch=272 <code>NC_004463</code>]), residing at location [http://www.ncbi.nlm.nih.gov/projects/sviewer/?id=NC_004463.1&v=5784144..5784239 <code>5784144-5784239</code>] within the genome, which is at the 5' side of the gene [http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=search&term=NC_004463%20hspb hspB].
 +
 
 +
===Links===
 +
* [http://2008.igem.org/Team:TUDelft/Temperature_design2 Extensive description of the design of this part.]
 +
* [http://rfam.sanger.ac.uk/family?entry=rose ROSE family in the Rfam database]
  
 
===References===
 
===References===

Latest revision as of 08:58, 28 October 2008

RNA thermometer (ROSE 27C)


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]


Design Notes

The designed sequence is based on a [http://en.wikipedia.org/wiki/Repression_of_heat_shock_gene_expression_(ROSE)_element ROSE] RNA thermometer retrieved from Bradyrhizobium japonicum USDA 110 . The temperature sensitive hairpin of this RNA thermometer (the one that contains the Shine Dalgarno (SD) sequence) is destabilized in order to get a lower switching temperature.

Incorporation of weak base pairs, bulge loops, and internal loops is used for the destabilization of the temperature sensitive hairpin, while ensuring that the secondary structure remains unaltered, and the distribution of the free energy within the temperature sensitive hairpin remains similar to the [http://2008.igem.org/Team:TUDelft/Temperature_analysis#Results characteristic free energy distribution] as found for 32 ROSE RNA thermometers. The dark blue line in the graph shows the found characteristic free energy distribution of the 32 ROSE RNA thermometers which have their switching point around 37°C. The light blue line shows the distribution of the free energy for this part at 27°C. This line is fitted to the trend line, so that the temperature sensitive hairpin of this part has the same stability, but at a lower temperature (27°C instead of 37°C). The assumption is that this will also cause the switching temperature to drop to 27°C.

The secondary structure of the part after ligation to a protein coding part, as predicted by [http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi RNAfold], is shown at the left. Notice that the 3' end, including the scar and the start codon, does not belong to the part. The blue region is the Shine Dalgarno sequence which is the ribosome binding site.

See our [http://2008.igem.org/Team:TUDelft/Temperature_design2 wiki] for an extended description of the design of this part.

BBa K115016.png

Source

RNA thermometer from Bradyrhizobium japonicum USDA 110 ([http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genome&Cmd=ShowDetailView&TermToSearch=272 NC_004463]), residing at location [http://www.ncbi.nlm.nih.gov/projects/sviewer/?id=NC_004463.1&v=5784144..5784239 5784144-5784239] within the genome, which is at the 5' side of the gene [http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=search&term=NC_004463%20hspb hspB].

Links

  • [http://2008.igem.org/Team:TUDelft/Temperature_design2 Extensive description of the design of this part.]
  • [http://rfam.sanger.ac.uk/family?entry=rose ROSE family in the Rfam database]

References