Excision of CMV from pIRES2-EGFP and following riligation of the backbone pIRES2-EGFP supplied by Clontech has been digested in AseI and NheI (Fig.1) in order to remove the constitutive CMV promoter and then the linearized backbone has been purified using the "Wizard Gel Clean Up System" by Promega.
The extremities of the linearized backbone have been blunted in order to allow its self ligation.
XL10-GOLD competent cells have been transformed with the products of ligation and then minipreps have been done.
The colonies have been checked by enzymatic digestion with NdeI and BamHI, the positives must show only one excised fragment of 600bp (Fig.2).

Excision of TraBox-CMV from pSEAP pSEAP has been double digested with EcorI and NotI in Buffer EcoRI plus BSA in 30ul total.
The digestion has been checked on agarose Gel 0.8% W/V.
The fragment corrensponding to the TraBox/CMVmin has been purified using the "Wizard Gel Clean Up System" by Promega.

Cloning TraBox-CMVmin in pCDNA3 using NotI- EcoRI sites: pCDNA3 has been previously cut in EcorI and NotI in order to obtain the linearized backbone ready for the cloning of TraBox- CMVmin.
Different Condition of ligation has been performed looking for the best efficiency.
The colonies obtained in this way have previously been screened by colony pcr and then checked by enzymatic digestion.
All the digested colonies were positive, the fragment excised by the EcorI/XhoI double digeston is the TraBox-CMVmin. (Fig.3)
One of the positives has been chosen and then amplified by trasformation in XL10-GOLD competent cells. The plasmidic DNA has been purified using a commercial Kit supplied by Promega.
The Plasmidic DNA has been subsequently digested in EcoRI and XhoI in order to obtain the same insert previously cloned provided by the XhoI sites.
The insert TraBox-CMVmin has been purified using the "Wizard Gel Clean Up System" by Promega.

Cloning TRABOXCMVmin in pIRES2-EGFP/CMV- using EcorI/XhoI sites in order to obtain pTraBOX-IRES-EGFP TraBox-CMVmin has to be cloned in the pIRES2-EGFP/CMV- previously digested in EcoRI XhoI.(Fig.4)
The linearized backbone has been purified using the "Wizard Gel Clean Up System" by Promega and then ligated with the TraBox- CMVmin as insert.
Different Condition of ligation has been performed looking for the best efficiency.
XL10-GOLD competent cells have been transformed with the products of ligation and then minipreps has been done.
The plasmidic DNA so obtained has been screened by enzymatic digestion using EcoRI and XhoI. The positives have to show the TraboxCMVmin excised in agarose gel electrophoresis separation (Fig5).
Colony N°2 and 4 has been chosen as positive and amplified in order to obtain more plasmidic DNA.

Cloning sBLA in pTraBox-IRES-EGFP sBLA has to be cloned in the pTRABOX-IRES-EGFP previously digested in EcoRI - BamHI.
The linearized backbone has been purified using the "Wizard Gel Clean Up System" by Promega and then ligated with the sBLA as insert.
Different Condition of ligation has been performed looking for the best efficiency.
XL10-GOLD competent cells have been transformed with the products of ligation and then minipreps has been done.
The plasmidic DNA so obtained has been screened by enzymatic digestion using EcoRI and BamHI.
The positives have to show the sBLA excised in agarose gel electrophoresis separation(Fig.6).
Colony N°4 and N°5 have been chosen as positive and amplified in order to obtain more plasmidic DNA.

Checking the final constructs pTRABOX-sBLA-IRES-EGFP In order to check the final constructs both the plamidic DNA obtained by the clone N°4 and 5 has been digested with: -   EcoRI-BamHI: sBLA has to be excised
-   EcoRI-XhoI: TraBoxCMVmin has to be excised
-   NdeI-BamHI: The construct has to be linearized
All the digestions have been checked in Gel electrophoresis separation on Agarose 1% W/V (Fig.7)

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.