Part:BBa_K1925003
CMV-tTA-PA signal-tetO U6
We improved a previous tet-off promoter (BBa_K1061013) for non-coding RNA expression. Non-coding RNA could be easily inserted through digestion and ligation. We used it to drive our shRNAs expression and it actually performs well (See our Result). tet-off response ability and leakness is also tested and shown in our Result. Though it is used for ncRNA transcription, it can also be used to express mRNA only with a attenuated activity.
Since there are abundant enzyme cutting sites in our tet-off U6, we failed to clone and paste the fragment into pSB1C3 to meet any RFC criteria. Therefore we submitted our original plasmid contains this system (pShuttle-tet-off-U6, pShuttle serves to help recombinant in adenovirus assembly) to iGEM Registry, and we upload the plasmid sequence to Addgene. Anyone can acquire it from http://www.addgene.org/vector-database/.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 620
Illegal XbaI site found at 629
Illegal PstI site found at 1373 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 620
Illegal NheI site found at 582
Illegal PstI site found at 1373 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 620
Illegal BglII site found at 600
Illegal BamHI site found at 1501
Illegal XhoI site found at 604 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 620
Illegal XbaI site found at 629
Illegal PstI site found at 1373 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 620
Illegal XbaI site found at 629
Illegal PstI site found at 1373
Illegal NgoMIV site found at 1246 - 1000COMPATIBLE WITH RFC[1000]
Results
We used 293T cell line to test system we constructed. we adopted GFP as the report gene, and this system behaves extremely good in 293T cell. Different dose of doxycycline is applied to cell, and after 24 hours GFP fluorescence is detected only in 0ug/ml group, though 0.01ug.ml group has very low background. Positive control cell is transfected with CMV-GFP compared to tet-off-U6-GFP in experimental groups. Since CMV performs better than U6 in expressing coding mRNA, GFP is brighter in Control than in 0 μg/ml group. Trace amounts of Dox significantly suppress expression without turnover. Cell density is alike among different groups.
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