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 B1, composed of the LasR-regulated promoter (P-LasR, BBa_R0079), an E.coli ribosome binding site (RBS, BBa_J15001) and a double terminator (BBa_B0015); 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 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 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. We thus sent B1 to be sequenced as well, and in meantime started working to construct it from the start again. 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.