Difference between revisions of "Part:BBa K2254520"
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H5-2 Toehold Switch is one of the synthetic toehold switch that is designed to detect the Hemagglutinin 5 (H5) RNA in influenza A virus. iGEM17 Hong Kong-CUHK team designed 3 toehold switches to detect the Hemagglutinin 5 (H5) gene (BBa_K2254510, BBa_K2254520, BBa_K2254530). | H5-2 Toehold Switch is one of the synthetic toehold switch that is designed to detect the Hemagglutinin 5 (H5) RNA in influenza A virus. iGEM17 Hong Kong-CUHK team designed 3 toehold switches to detect the Hemagglutinin 5 (H5) gene (BBa_K2254510, BBa_K2254520, BBa_K2254530). | ||
| + | Switch plasmid is transformed either with its respective trigger or empty plasmid into E. coli BL21 (DE3) cells. Single colony of each set-up was picked to grow overnight culture. Expression of reporter mRFP was done by shaking culture for 6 hours after 1% inoculation when log phase was reached. After harvesting the cells and determining the OD600, cells were washed with PBS buffer, concentrated 10x and then aliquoted in 96-well plates to determine the fluorescent signal by exciting at 584nm and measuring at 607nm. Final fluorescent signal were normalized by OD600 to give the final relative fluorescent unit (R.F.U). Below graph showed the result of H5-1, H5-2 and H5-3 switches (BBa_K2254510, BBa_K2254520, BBa_K2254530). | ||
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| + | [[File:CUHK_H5.png|400px]] | ||
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Revision as of 19:07, 29 October 2017
H5-2 Toehold Switch
H5-2 Toehold Switch is one of the synthetic toehold switch that is designed to detect the Hemagglutinin 5 (H5) RNA in influenza A virus. iGEM17 Hong Kong-CUHK team designed 3 toehold switches to detect the Hemagglutinin 5 (H5) gene (BBa_K2254510, BBa_K2254520, BBa_K2254530).
Switch plasmid is transformed either with its respective trigger or empty plasmid into E. coli BL21 (DE3) cells. Single colony of each set-up was picked to grow overnight culture. Expression of reporter mRFP was done by shaking culture for 6 hours after 1% inoculation when log phase was reached. After harvesting the cells and determining the OD600, cells were washed with PBS buffer, concentrated 10x and then aliquoted in 96-well plates to determine the fluorescent signal by exciting at 584nm and measuring at 607nm. Final fluorescent signal were normalized by OD600 to give the final relative fluorescent unit (R.F.U). Below graph showed the result of H5-1, H5-2 and H5-3 switches (BBa_K2254510, BBa_K2254520, BBa_K2254530).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 120
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 680
Illegal AgeI site found at 792 - 1000COMPATIBLE WITH RFC[1000]