Part:BBa_K3956016:Experience

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Applications of BBa_K3956016

To create a plasmid that could be cloned in yeast cells, several linear fragments had to be prepared, as the desired scl2 construct (BBa_K3956014) by itself was too complicated to be prepared by custom synthesis. Throughout the optimization of these fragment designs with 80-bp overhangs to each other, the linearized plasmid backbone consisting of the pRS4116 plasmid (with the lacZalpha and f1ori regions removed) was kept constant. Figure 1.1 compares the lengths of the amplified, linearized backbone to the full pRS4116 plasmid, with the linearized fragment in lane 1 slightly shorter, implying successful PCR. The presence of this linear backbone fragment was further verified after a DpnI digest was conducted to remove any methylated DNA still part of a circular plasmid backbone (Figure 1.2). As the PCR product only has one visible band around the desired length, 3754 bp, in comparison to the same concentration of plasmid backbone, it can be concluded that the DpnI was successful.

Although we were able to verify that the construct had been correctly inserted into the backbone and plated on appropriate selective media by colony PCR verification, the protein expression was not able to be verified within the time constraints of the competition. We want to emphasize that this sequence may still be successful, but its efficiency was unable to be demonstrated within the time span that CCA_San_Diego had in the lab. For future teams, we advise testing construct designs with successful expression with and without the sequence to compare expression levels.

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