Part:BBa_K838000

Mammalian LovTAP-VP16

LovTAP-VP16 is a light-sensitive protein that binds to DNA. The protein is a fusion between a Lov light activated domain, a TRP repressor, an NLS/ linker domain and a VP16 activator domain. As it contains a VP16 activational domain, it's able to act as a transcriptional activator when it is bound to a TRP promoter site.

The lov domain clashes with the TRP repressor domain and prevents binding before the protein is exposed to blue light. After light activation the Lov domain goes through a comformational change and the TRP repressor domain of the protein can bind DNA.

Source

This part is an improvement of part BBa_K191006 designed by the 2009 EPF-Lausanne team (LovTAP, a light-sensitive repressor expressed in bacteria).

Usage and Biology

1) Cloning:

First clone the part into a mammalian expression vector such as [http://products.invitrogen.com/ivgn/product/V79020?ICID=search-product pcDNA3.1(+)] or [http://products.invitrogen.com/ivgn/product/V04450 pCEP4]. These are the two main expression vectors we used.

Any promoters already present must be removed since we want only want expression drive by LovTAP-VP16 and not constitutive expression. The part must have a polyA tail at the end as well.

LovTAP Mammalian + dsRed readout BBa K838001

2) Transfection Co-transfect the combination of LovTAP-Vp16 and readout in mammalian cells!

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 274
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 772
  Illegal XhoI site found at 817
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 274
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 274
  
• 1000
  COMPATIBLE WITH RFC[1000]