Team:UNITS Trieste/Project
From 2011.igem.org
pTraBox
P65-TraR
AHL Sensible Eukaryotic Switch
We decided to test both pTraBox-SEAP and p65-TraR (Neddermann P. et al., 2003), kindly provided by Dr. R. Cortese's group, using SEAP (Secreted alkaline phosphatase) as reporter gene, detected with the Great Escape Chemiluminescent assay kit (Clontech).
In our final system we aim to have the presence of both the OXOC8 and the OXOC12 but the eukaryotic cell has to be sensible only to OXOC8.
On this basis the assay was performed in order to test the efficiency of this inducible promoter after the induction with OXOC8 and the response to OXOC12 as unspecific ligand.
AHL has to be dissolved in a organic solvent as Ethyl-Acetate in order to prevent the lactonolysis that will occur in prolonged exposure to aqueous conditions.
2x105 cells for Hela were placed in 35mm culture dishes and transfected using the Fugene HD transfection reagent (Promega). For each transfection 2ug of DNA were transfected.
For all the experimental conditions that we tested, were performed biological triplicates and experimental triplicates.
Figure 1. SEAP activity12 hours after transfection.
2x105 cells for Hela were placed in 35mm culture dishes and transfected using the Fugene HD transfection reagent (Promega). For each transfection 1ug of transactivator plasmid (P65-TraR) and 1ug of pTraR-SEAP reporter were transfected. We decided also to test the basal activity of SEAP under the control of TraBox-CMVmin, in order to achieve this goal hela cells were transfected with 1ug of pTraR-SEAP and 1ug of pCDNA3.
After 6 hours 20uM of AHLs (OXOC8 and OXOC12 separately) were added to cell culture medium and 12 hours after the addition of ligands the medium was collected and the activity of SEAP was measured.
Hela WT were treated with a corresponding amount of Ethyl Acetate + OXOC8.
Figure 2. SEAP activity 24 hours after transfection.
2x105 cells for Hela were placed in 35mm culture dishes and transfected using the Fugene HD transfection reagent (Promega). For each transfection 1ug of transactivator plasmid (P65-TraR) and 1ug of pTraR-SEAP reporter were transfected. We decided also to test the basal activity of SEAP under the control of TraBox-CMVmin, in order to achieve this goal hela cells were transfected with 1ug of pTraR-SEAP and 1ug of pCDNA3.
After 6 hours 20uM of AHLs (OXOC8 and OXOC12 separately) were added to cell culture medium and 24 hours after the addition of ligands the medium was collected and the activity of SEAP was measured.
Hela WT were treated with a corresponding amount of Ethyl Acetate + OXOC8.
Figure 3. Luciferase activity12 hours after transfection. 2x105 cells for Hela were placed in 35mm culture dishes and transfected using the Fugene HD transfection reagent (Promega). For each transfection 1ug of Luciferase reporter plasmid and 1ug of pCDNA3 reporter were transfected as positive control of trasnfection. Cells were treated with 20 uM OXOC8 6 hours after trasnfection.