Difference between revisions of "Part:BBa K115017:Design"
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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. | 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 orange line shows the distribution of the free energy for this part at | + | 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 orange line shows the distribution of the free energy for this part at 32°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 (32°C instead of 37°C). The assumption is that this will also cause the switching temperature to drop to 32°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. | 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. | ||
Latest revision as of 13:33, 28 November 2008
RNA thermometer (ROSE 32C)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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 orange line shows the distribution of the free energy for this part at 32°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 (32°C instead of 37°C). The assumption is that this will also cause the switching temperature to drop to 32°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.
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]