Part:BBa_K2748001

rtTA-advanced (reverse tet-controlled transactivator)

Biology and Usage

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.

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.

Design Considerations

The nucleotide sequence was obtained from our host lab. Biobrick prefix and suffix was added by PCR using the following primers,

rtTA-prefix: 5’ cggaattcgcggccgcttctagatgtcTagGctggacaagagcaaagTC 3’

rtTA-suffix: 5’ AActgcagcggccgctactagtattacccggggagcatgtcaag

3’ 

and ligated onto the pSB1C3 plasmid backbone obtained from digestion of pSB1C3-lacI-RFP(Part:BBa_J04450). The sequence was comfirmed by Sanger sequencing by IGE Biotechnology LTD using VF2 as the forward primer.

Note that this promoter has basal leaky transcription

Also note that this promoter needs to be used in stable cell lines expressing rtTA or in cells co-transfected with expression vector for rtTA.

Results

For more details, please check out our [http://2018.igem.org/Team:SYSU-CHINA#/Demonstrate result page]!

In iGEM 2018, SYSU-CHINA attempted to develop a reversible safe switch for CAR T therapy based on the tet-inducible CMV promoter and U24 protein of Human Herpesvirus 6. Since it is the major part of our project, we conducted extensive research on U24 and obtained valuable results..

In order to determine the optimal concentration of dox for induction, we collected and analyzed fluorescence data and performed western blot analysis.

Interestingly, although the predicted molecular weight of U24 protein is approximately 10kDa, two bands were detected at 20kDa, consistent with previous research (Sullivan and Coscoy, 2010). This suggested that U24 undergoes extensive post-translational modifications. However, the types and functions of these modifications remain unknown.

Figure 2: Fluorescence and western blot analysis of U24 expression under different concentration of doxycycline

In order to determine the expression time course after adding dox, equal amount of HEK293T cells were seeded in a 6-well plate and transfected with ptetON-GFP-T2A-U24. Dox was added at the same time in all wells and equal number of cells in each well were harvested at the indicated time for western blot analysis. The result was analyzed with ImageJ and the amount of protein of interest was normalized using β-actin as internal controls.

Figure 3: Time course of U24 expression

References

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 409
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 347
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 165