Team:UNITS Trieste/Notebook

Our lab work starts off with the arrival of the two plasmids pcD/p65NTraR and pSEAP-(tra box)(1-7) (Neddermann P. et al., 2003), kindly provided by Dr. R. Cortese’s group, and of the commercial vector pIRES2-EGFP (Clontech), in which we are going to subclone all the parts needed to engineer our eukaryotic cells.
Different competent E.coli strains (XL10-Gold and DH5-alpha) have been transformed with the above mentioned plasmids and selected with appropriate culture conditions (p65NTraR and pSEAP grow on ampicillin, pIRES2-EGFP grows on kanamicin).
Subsequently the amplified plasmids have been purified following a regular Miniprep protocol.
Concurrently, we have designed and ordered two oligos needed in order to remove the CMV immediate early promoter (CMVie) from pIRES2-EGFP, and to allow the correct insertion of the secreted mammalian beta-lactamase (sBLA) coding sequence into the vector.
We then started a series of enzymatic digestions in order to extrude CMVie from pIRES2-EGFP and just take its backbone, and to isolate sBLA from pSEAP.
In the meantime the plasmid pWW1015 we requested from Dr. M. Fussenegger arrived and was immediatedly amplified through transformation of DH5-alpha bacteria, while we also ordered the sequencing of the p65NTraR plasmid (BMR Genomics).

The oligos we requested the earlier week arrived, and we proceeded with the annealing protocol for the two pairs of complementary strands.
Due to the irregular running pattern pSEAP displayed on agarose gel, we set out to do several control digestions in order to determine its integrity and we also sent a DNA sample to sequence its multiple cloning site (MCS).
Meanwhile we also started the prokaryotic part of this work by transforming competent bacteria with all the Biobricks needed in this side of the project, and by PCR-amplifying the TraR-regulated promoter, TraI gene and TraR gene sequences from A.tumefaciens genomic DNA.
This Tra family has been cloned into pBluescript and then used to transform bacteria on Xgal-containing Petri dishes. Finally, a colony PCR has been carried out to further amplify the Tra family DNA sequences.
Moreover we transformed new bacteria in order to amplify a plasmid containing the enzyme beta-glucosidase (bGluc) we received from Dr. C. French.

We obtained pIRES2-EGFP without CMVie but could not ligate our oligo inside it, so we devised new ligation strategies to amplify our backbone, removing all the unnecessary DNA sequences.
We also started working on some of the constructs needed to engineer our bacterial strains, namely: the bacterial construct A0, composed of a TraR-regulated promoter (P-TraR) followed by a beta-glucosidase (BBa_K392008), an E.coli ribosome binding site (RBS, BBa_J15001) and a double terminator (BBa_B0015); the bacterial construct B1, identical to A0 but under the transcriptional control of a LasR-regulated promoter (P-LasR, BBa_R0079); the bacterial construct B2, consisting of a constitutive promoter (BBa_J23119), an RBS (BBa_B0034), a LasR activator (BBa_C0179) and a terminator; and the bacterial construct B3, made of P-LasR, an RBS, the TraI gene and a terminator.

We finally obtained and amplified pIRES2-EGFP without CMVie and without the gene Rac2, that we discovered was cloned inside the MCS.
We then proceeded to amplify an aliquot of pSEAP of which we received the full sequence, in order to transfect HeLa cells and assay it’s activity.
We amplified and ligated TraI with BBa_B0015 in order to subsequently complete the bacterial construct B3. The construction of A0 and B2 is approaching completion as well, while we finished working on constrct B1: we’ll now need to verify the correctness of its sequence.
Meanwhile we also started working on our bacterial construct A2 by ligating the previously amplified P-TraR into BBa_K081016 (LasI comprising an RBS and a terminator up- and downstream, respectively).

We sent the sequences to have the secreted beta-lactamase (sBLA) gene by synthesis, comprising all the restriction sites needed to clone it into our vectors. It should be here next week
Also, we tested the activity of pSEAP after co-transfection with p65-TraR into HeLa cells. To assay this we employed the Great EscAPe secreted alkaline phosphatase detection kit (Clontech), obtaining promising results confirming the efficiency of the Tra box-CMVminimal promoter sequence. We then proceeded to isolate this DNA sequence and ligate it into pIRES2-EGFP (lacking its constitutive CMV promoter), through a series of sequential cloning steps.
The bacterial constructs A2 and B2 have been completed and sent to sequence, while B1 was control-digested but found not to be of the expected dimensions. The same problem occurred with A0, probably due to the beta-glucosidase we used for both the constructs. We thus sent A0 and B1 to be sequenced as well, and in meantime started working to construct it again from the start. The construction of B3 proceeded with the ligation of the TraI/terminator segment with P-LasR/RBS and was followed by the transformation of competent bacteria to subsequently verify the possible completion of the construct.

The Tra box-CMVmin sequence was cloned into pIRES2-EGFP (without CMV), so we wanted to repeat the earlier week’s experiment and see if, following the correct AHL signal, cells would express EGFP. Curiously only a light fluorescence was detected, either due to an error during the cloning steps, or because of the low translation efficiency of the IRES sequence.
Meanwhile the sBLA we ordered by synthesis arrived, so we set out to amplify this construct in order to clone it in an appropriate vector and assay it’s activity.
The results from last week’s sequencing confirmed the successful completion of the bacterial constructs A2 and B2. The sequencing of A0 and B1, on the other hand, did not give the same results. On the contrary, we found a few discrepancies between BBa_K392008’s sequence and that which we were making reference to till now, so the work to re-construct A0 and B1 is still ongoing.
Finally, the construct B3 was completed, controlled, and then ligated into B2.

The synthetic sBLA construct was cloned into pSEAP downstream of the Tra box-CMVmin sequence. This plasmid was then tested after transfection into HeLa cells and successive AHL induction in order to verify the secretion of the enzyme. Early analyses showed negative results, probably due to the low levels of protein produced or to errors during the cloning design. We then set out to do more clonings into other vectors in order to further test sBLA activity. In particular we cloned the synthetic sBLA into pIRES2-EGFP (either with and without CMV), and we also amplified pWW1015 to clone its sBLA into pcDNA3.
Meanwhile the bacterial constructs A0 and B1 have been completed and assayed for their activity in response to the appropriate AHL signals (OC8 and OC12, respectively). This assay, which gave good results, showed that these constructs coding for an AHL-inducible beta-glucosidase allow bacteria to survive in a glucose free medium supplied with cellobiose alone. Finally, we also tested our bacteria for the production of AHL on thin layer chromatography.