Difference between revisions of "Part:BBa K2254030"
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PB2-3 Toehold Switch is one of the synthetic toehold switch that is designed to detect the polymerase basic protein 2 (PB2) RNA in influenza A virus. iGEM17 Hong Kong-CUHK team designed 3 toehold switches to detect the Polymerase basic protein 2 (PB2) gene (BBa_K2254010, BBa_K2254020, BBa_K2254030). | PB2-3 Toehold Switch is one of the synthetic toehold switch that is designed to detect the polymerase basic protein 2 (PB2) RNA in influenza A virus. iGEM17 Hong Kong-CUHK team designed 3 toehold switches to detect the Polymerase basic protein 2 (PB2) gene (BBa_K2254010, BBa_K2254020, BBa_K2254030). | ||
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| + | 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 PB2-1, PB2-2 and PB2-3 switches (BBa_K2254010, BBa_K2254020, BBa_K2254030). | ||
| + | For this toehold switch, we obtained increased amount of RFP in co-transformed culture. Noted that the difference was not statistically significant in our trial. The difference in fluorescent signal was only observed at 4-6 hours. No difference was observed in colonies. This switch is not stable. | ||
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| + | [[File:CUHK_PB2.png|400px]] | ||
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Latest revision as of 07:29, 14 June 2018
PB2-3 Toehold Switch
PB2-3 Toehold Switch is one of the synthetic toehold switch that is designed to detect the polymerase basic protein 2 (PB2) RNA in influenza A virus. iGEM17 Hong Kong-CUHK team designed 3 toehold switches to detect the Polymerase basic protein 2 (PB2) gene (BBa_K2254010, BBa_K2254020, BBa_K2254030).
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 PB2-1, PB2-2 and PB2-3 switches (BBa_K2254010, BBa_K2254020, BBa_K2254030).
For this toehold switch, we obtained increased amount of RFP in co-transformed culture. Noted that the difference was not statistically significant in our trial. The difference in fluorescent signal was only observed at 4-6 hours. No difference was observed in colonies. This switch is not stable.
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]