Team:UNITS Trieste/Notebook

1st Week

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).

2nd Week

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 TraI 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.