Part:BBa_K4151015

IRES

Internal ribosome entry site(IRES) is a joint gene engineering biobrick that recruits ribosomes to the sequence and translate without cap manners. We decided to place the IRES Biobrick before the sequence that codes the rtTA complex, which is needed to bind to TetO to drive the sequence (TetO is also known as Tet-on system promoter). A drawback of IRES sequence is that the expression of the downstream protein would be relatively lower than the specific promoter. However, it is advantageous to our project since several papers indicate that rtTA is toxic to immune cells. Our design can reduce rtTA expression when doxycycline is absent. However, while doxycycline is added, the Tet-on promoter construct will drive the entire sequence and result in a positive cycle that increases the rtTA expression and the sequence driven by the TetO promoter at the same time. To sum up, our system can control both rtTA and target protein at a deficient background level when tetracycline is not added and increase the amount of rtTA and AID in a short time when tetracycline is added. On the contrary, the traditional Tet-on system would have a higher background level of rtTA and target protein when tetracycline is not added.

Reference

Markusic D, Oude-Elferink R, Das AT, Berkhout B, Seppen J. Comparison of single regulated lentiviral vectors with rtTA expression driven by an autoregulatory loop or a constitutive promoter. Nucleic Acids Res. 2005 Apr 4;33(6):e63. doi: 10.1093/nar/gni062. PMID: 15809225; PMCID: PMC1074399.

Morimoto M, Kopan R. rtTA toxicity limits the usefulness of the SP-C-rtTA transgenic mouse. Dev Biol. 2009 Jan 1;325(1):171-8. doi: 10.1016/j.ydbio.2008.10.013. Epub 2008 Nov 1. PMID: 19013447; PMCID: PMC2955437.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]