This year, SYSU-CHINA developed a reversible safe switch for CAR T therapy based on the tet-inducible CMV promoter and U24 protein of
Human Herpesvirus 6. To our knowledge, tet-inducible transcription system is a widely-used inducible expression system in mammalian
cell lines, yet few available part is in the registry, left alone limited information for those part. Aiming to benefit the iGEM
community, we standardized both the tet-ON promoter and the coding region of reverse tet-responsive transactivator in our project by
cloning the sequences into the standard plasmid backbone pSB1C3, and submitted them to the iGEM registry. To ensure the parts'
quality, we sequenced the part using VF2 as primer before submitting the plasmids. In addition, we provided first-hand data on the
tet-ON promoter and rtTA. Hopefully, teams in the future that are ambitious to conduct projects requiring inducible expression will
find our parts useful.
Tet-ON promoter (BBa_K2748002, Improved part for BBa_K368001)
Tet-ON promoter is the promoter sequence in the Tet-inducible transcription system (tet-ON system).
Tet-ON system(Gossen et al., 1995) is a inducible transcription system widely used in mammalian
cells. The tet-ON system utilizes the sequence-specific DNA binding property of tet repressor protein (tetR) from Escherichia coli in
the presence of tet or dox, and consists of two parts: The Tet-inducible CMV promoter (tet-ON promoter) and reverse tetracycline-
controlled transactivator (rtTA). The tet-ON promoter consists of tandem tetracycline-responsive elements (TRE) followed by a minimal
CMV promoter. The rtTA protein comprises of reverse-tetR (rtetR, mutant of tetR) and activation domains from herpes simplex virus
VP16. When tet or dox is added, the rtTA binds to TRE, and VP16 domain will recruit factors of RNA polymerase II to initiate
transcription. In the absence of tet or dox, the rtTA detaches from tetON promoter, and thus no transcription.
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Reverse tetracycline-controlled transactivator (rtTA) (BBa_K2748001)
rtTA is the transactivator in the Tet-inducible transcription system (tet-ON system) that selectively binds to tet-ON promoter in the
presence of tetracycline or doxycycline and activate transcription. By submitting this to the registry, we aims to benefit the
community that teams in the future can establish their own stable cell lines for for tet-inducible expression, or integrate it into
vectors containing tet-ON promoter (like we did!) for tet-inducible expression of gene of interest.
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