Part:BBa_K838001

LovTAP readout This part needs to be used with part BBa_K838000, which is LovTAP-VP16.

The part consists of a TRP repressor and DsRed. The LovTAP-Vp16 protein attaches to the this readout and promotes transcription of the DsRed mRNA.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 424
  Illegal SpeI site found at 295
  Illegal SpeI site found at 303
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 424
  Illegal SpeI site found at 295
  Illegal SpeI site found at 303
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 424
  Illegal BglII site found at 381
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 424
  Illegal SpeI site found at 295
  Illegal SpeI site found at 303
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 424
  Illegal SpeI site found at 295
  Illegal SpeI site found at 303
  Illegal NgoMIV site found at 7
  
• 1000
  COMPATIBLE WITH RFC[1000]

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.

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

3) Illumination Continuous illumination with blue light (468 nm) is recommended for the LovTAP protein. To build a set up like the one we used, take a look at our [http://2012.igem.org/Team:EPF-Lausanne/Bioreactor Bioreactor assembly page], where we explain what components you need, how to assemble them and how to program your illumination device.

4) Test the expression Since this readout system is built to express dsRed, a fluorescent protein, difference in fluorescence between cells in light or dark state can be measured using flow cytometry. This system still needs experimental optimisation, figure out how long the cells need to be illuminated, if they need a resting time in the dark, when exactly the dsRed is most expressed etc. Look at the Experience page and our [http://2012.igem.org/Team:EPF-Lausanne wiki] for more infos!