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.

This plasmid hosts an OC8HLA inducible promoter (BBa_K553002). Once OC8 HLA has bound the TraR trans-activator the cellobiosidase (Bba_K392008) transcription is activated thus the bacteria can transform the cellobiose in glucose and use it as a source of energy.
The plasmid provides also to generate constitutively RFP and host a kanamycin resistance.
TraR and Prom TraI are new biobrick obtained by Agrobacterium Tumefacens

To build this plasmid we used the following parts:
-Promoter Tra I regulated from A. Tumefaciens (New!) - 151 bp
-RBS BBa_J15001 + Glucosidase BBa_K392008 composite Biobrick - 1681 bp
-Terminator BBa_B0015 - 129 bp
To learn more about the extraction of PromTraI, see plasmid PromTraI – RBS – LasI – terminator

The double terminator BioBrick (BBa_B0015) has been resuspended and then amplified through transformation into DH5α competent cells. The plasmid was then extracted and digested (EcoRI/XbaI). The RFC10 compatible RBS-Glucosidase plasmid (BBa_J15001 + BBa_K392008), a gift from Dr. C. French (University of Edinburgh), was transformed into DH5α, extracted and digested (EcoRI/SpeI). The glucosidase was then ligated inside the BBa_B0015 vector and transformed into DH5α. The transformed colonies were screened with colony PCR (primers: vf2 and Vr), using the following protocol: 93° 5’ | 30x(95° 30” | 50° 30” | 72° 60”) | 72° 7’ | 4° ∞

The plasmid was extracted from the positive colonies and then digested with EcoRI/XbaI in order to linearize it and ligate it to PromTraR, previously digested with EcoRI/SpeI.
DH5α were transformed with the ligation product and the resulting colonies were PCR-screened (primers: vf2 and Vr). The first screening used the following protocol: 93° 5’ | 30x(95° 30” | 50° 30” | 72° 60”) | 72° 7’ | 4° ∞ There was no amplification: probably the sequence is too long.
An alternative protocol, which made use of different and more specific primers flanking the PromTraR sequence (primers: PromTra Fw and PromTra Rev), was employed: 93° 5’ | 30x(95° 30” | 65° 30” | 72° 30”) | 72° 7’ | 4° ∞ The second screening identified some positive colonies, giving a PromTraR amplicon of less than 200 bp. These positive colonies were then amplified to extract suitable amounts of the plasmid. The control digestion of the complete plasmid with EcoRI/PstI showed something unexpected, so we sent the DNA to sequence and in the meantime we started to build again our glucosidase construct from the beginning. We restarted from a different colony of BBa_J15001 and extracted its plasmid. We obtained a clean EcoRI/SpeI cut and ligated it to the glucosidase inside the vector containing the terminator with different strategies. For each ligation, a transformation and a colony PCR have been made (primers: vf2 and Vr).
The last colony PCR finally showed some positive colonies. The protocol used was different, in order to bypass the problem of the amplicon length: 93° 5’ | 30x(95° 30” | 50° 30” | 72° 90”) | 72° 7’ | 4° ∞ The plasmid was exracted from one positive colony, then digested with EcoRI/SpeI. Finally it was ligated to the EcoRI/SpeI-cut PromTraR.
DH5α were subsequently transformed and PCR-screened with different protocols: one amplifying the whole sequence and another specific for the PromTraR sequence. The former gave no result, but the latter revealed the incorporation of the promoter inside the glucosidase vector.
Some of the positive colonies were then chosen to amplify and extract the plasmid. We quality-controlled the plasmid with an EcoRI/PstI digestion, which showed as expected the construct at about 2000 bp and the plasmid backbone at 3000 bp. This DNA was sent to sequence and the results have been analyzed and compared with the previous construct we had made. The composite construct is correct but we found out some mutations in the glucosidase sequence.

To complete our construct, the BBa_I13521 PTet mRFP has been resuspended, transformed, extracted and digested with XbaI/PstI. The fragment has been purified from gel and ligated into our SpeI/PstI-digested construct, and then transformed into DH5α.

The plasmid obtained from the positive colonies, which were visibly red for the expression of mRFP, was digested with EcoRI/PstI and then cloned into the kanamycine-resistant pSB1K3 expression vector.
The positive red colonies growing from this last ligation contained our completed construct in the new plasmid.