Collaboration - Team Ui Oslo

Early on we encountered some trouble with basic cloning procedures, like mistakes in primer design, suboptimal ligation protocols and poorly chosen E. coli strains. When team Oslo approached us for help with the cloning of two constructs, which they had some problems with and didn’t have the time to redo, we were happy to help. The idea was that we’d receive a PCR-product of the parts, do the cloning of the parts into the shipping vector and then send that off to the parts-registry, as well as back to them for characterization. Unfortunately customes held up the shipping for so long that sending the parts back for characterization was out of the question. Also the primers used to amplify the part before restriction and ligation were constructed without keeping in mind, that the terminal restriction sites of both the BioBrick-prefix and BioBrick-suffix have to have at least 1 additional nucleotide at either sides for any activity of the restriction enzymes. That is true for all engineered PstI and EcoRI enzymes from NEB and Thermo Scientific. Thus the usual protocol would not work.

We attempted cloning using the XbaI- and SpeI-sites. That had the obvious disadvantage of possibly recombining the wrong way around or the vector religating without an insert, since XbaI and SpeI share the same overhang (namely CTAG). However, if either of these unfortunate outcomes was to occur, the plasmid could not be digested with XbaI and SpeI anymore. That opened up an easy way to check for the correct insertion. This cloning was done and 16 colonies were picked. However none was tested positive using SpeI-digestion, which is to say, none was linearized after incubation with SpeI in correct buffer. As a last resort attempt, we tried TA-cloning. The DNA was amplified with a Taq-polymerase, which adds one more A at the 3’ end of the amplified DNA-fragment. Thus, using a linearized backbone with a T-overhang (pGEM), cloning should have been possible. From that plasmid subcloning into pSB1C3 could occur. Unfortunately this attempt did not produce any colonies after transformation. Sadly at this point it was too late for any other attempts and all we could do was to report our efforts and the encountered problems back to team Oslo.