Difference between revisions of "Part:BBa K3281011"
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Our project aims at expressing AID in hybridoma cells. Since AID is a deaminase that would induce point mutation, the expression of AID should be tightly regulated. In order to achieve this purpose, we incorporate Tet-on system, an inducible protein expression system, into our design. When tetracycline is added, it combined with rtTA (reverse tetracycline-controlled transactivator) to form a rtTA-tetracycline complex, which can recognize and attach to TetO sequence and express our target protein. By this approach, our construct is able to regulate the amount of AID expressed. However, excessive rtTA may pose harm to cells, so people can’t turn off the tet-on system too late. | Our project aims at expressing AID in hybridoma cells. Since AID is a deaminase that would induce point mutation, the expression of AID should be tightly regulated. In order to achieve this purpose, we incorporate Tet-on system, an inducible protein expression system, into our design. When tetracycline is added, it combined with rtTA (reverse tetracycline-controlled transactivator) to form a rtTA-tetracycline complex, which can recognize and attach to TetO sequence and express our target protein. By this approach, our construct is able to regulate the amount of AID expressed. However, excessive rtTA may pose harm to cells, so people can’t turn off the tet-on system too late. | ||
| − | =References= | + | ===References=== |
Stieger K, Belbellaa B, Le Guiner C, Moullier P, Rolling F. In vivo gene regulation using tetracycline-regulatable systems. Adv Drug Deliv Rev. 2009 Jul 2;61(7-8):527-41. doi: 10.1016/j.addr.2008.12.016. Epub 2009 Apr 23. PMID: 19394373; PMCID: PMC7103297. | Stieger K, Belbellaa B, Le Guiner C, Moullier P, Rolling F. In vivo gene regulation using tetracycline-regulatable systems. Adv Drug Deliv Rev. 2009 Jul 2;61(7-8):527-41. doi: 10.1016/j.addr.2008.12.016. Epub 2009 Apr 23. PMID: 19394373; PMCID: PMC7103297. | ||
Lai, WC., Sun, H.S. & Shieh, JC. Establishment of tetracycline-regulated bimolecular fluorescence complementation assay to detect protein-protein interactions in Candida albicans. Sci Rep 10, 2936 (2020). https://doi.org/10.1038/s41598-020-59891-7 | Lai, WC., Sun, H.S. & Shieh, JC. Establishment of tetracycline-regulated bimolecular fluorescence complementation assay to detect protein-protein interactions in Candida albicans. Sci Rep 10, 2936 (2020). https://doi.org/10.1038/s41598-020-59891-7 | ||
Latest revision as of 06:48, 10 October 2022
TRE Promoter with Tet operators
This is TRE promoter with Tet operators. This is useful for driving reporter transcription in Tet-on pathways, such as the TANGO assay. In Tet-on pathways, release of tTA will result in downstream transcription from this promoter.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Contribution : iGEM22_CSMU_Taiwan
Our project aims at expressing AID in hybridoma cells. Since AID is a deaminase that would induce point mutation, the expression of AID should be tightly regulated. In order to achieve this purpose, we incorporate Tet-on system, an inducible protein expression system, into our design. When tetracycline is added, it combined with rtTA (reverse tetracycline-controlled transactivator) to form a rtTA-tetracycline complex, which can recognize and attach to TetO sequence and express our target protein. By this approach, our construct is able to regulate the amount of AID expressed. However, excessive rtTA may pose harm to cells, so people can’t turn off the tet-on system too late.
References
Stieger K, Belbellaa B, Le Guiner C, Moullier P, Rolling F. In vivo gene regulation using tetracycline-regulatable systems. Adv Drug Deliv Rev. 2009 Jul 2;61(7-8):527-41. doi: 10.1016/j.addr.2008.12.016. Epub 2009 Apr 23. PMID: 19394373; PMCID: PMC7103297.
Lai, WC., Sun, H.S. & Shieh, JC. Establishment of tetracycline-regulated bimolecular fluorescence complementation assay to detect protein-protein interactions in Candida albicans. Sci Rep 10, 2936 (2020). https://doi.org/10.1038/s41598-020-59891-7