Difference between revisions of "Part:BBa K2254999"
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<partinfo>BBa_K2254999 short</partinfo> | <partinfo>BBa_K2254999 short</partinfo> | ||
| − | This is an improved version of previous biobrick BBa_K1728019. The design of original biobrick aims to detect spermine N1-acetyltransferase (SAT) mRNA , which is a biomarker of oral cancer. We improved the biobrick by converting the luciferase reporter gene to mRFP reporter gene, which can be visualized by naked eyes to allow on-site detection. We also demonstrated that it can detect SAT transcript. | + | This is an improved version of previous biobrick BBa_K1728019. The design of original biobrick aims to detect spermine N1-acetyltransferase (SAT) mRNA , which is a biomarker of oral cancer. We improved the biobrick by converting the luciferase reporter gene to mRFP reporter gene, which can be visualized by naked eyes to allow on-site detection. We also demonstrated that it can detect SAT transcript. In contrast, the expression of reporter in the original BBa_K1728019 was lowered by co-transforming with trigger RNA. |
| − | 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. IPTG- induced 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 fluorescent signal of SAT switch when transformed with or without trigger. | + | 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. IPTG- induced 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 fluorescent signal of SAT switch when transformed with or without trigger. For this toehold switch, we can see increased amount of RFP is produced when the respective trigger is together co-transformed. |
| − | [[File:CUHK_satresult|400px]] | + | [[File:CUHK_satresult.jpg|400px]] |
| + | ===Reference=== | ||
| + | Original part: https://parts.igem.org/Part:BBa_K1728019 | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
| + | <br> | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
Latest revision as of 14:19, 1 November 2017
SAT toehold swicth
This is an improved version of previous biobrick BBa_K1728019. The design of original biobrick aims to detect spermine N1-acetyltransferase (SAT) mRNA , which is a biomarker of oral cancer. We improved the biobrick by converting the luciferase reporter gene to mRFP reporter gene, which can be visualized by naked eyes to allow on-site detection. We also demonstrated that it can detect SAT transcript. In contrast, the expression of reporter in the original BBa_K1728019 was lowered by co-transforming with trigger RNA.
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. IPTG- induced 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 fluorescent signal of SAT switch when transformed with or without trigger. For this toehold switch, we can see increased amount of RFP is produced when the respective trigger is together co-transformed.
Reference
Original part: https://parts.igem.org/Part:BBa_K1728019 Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 105
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 665
Illegal AgeI site found at 777 - 1000COMPATIBLE WITH RFC[1000]