Part:BBa_K5490017:Design

The NFAT construct was kindly provided by Benčina M, with the goal of studying its translocation to the nucleus after ionophore stimulation. For detection, we used an immunohistochemical approach targeting the Myc-tag epitope, which was fused to the N-terminus of the NFAT construct. However, the antibody against the Myc-tag epitope generated significant background noise. While it had some affinity for its target, it also bound nonspecifically to other molecules, leading to an inconclusive image under the microscope. Despite this issue, we confirmed the expression of the synthetic protein via Western blot, which motivated us to explore alternatives. Specifically, we decided to add a Flag tag in front of the Myc-tag epitope, leading us to try two different cloning strategies to achieve this.

First, we attempted to fuse the Flag tag using subcloning. In this approach, we extracted the insert and blunt-ended one side of both the vector and the insert while keeping the other side sticky to maintain directionality during the ligation step. Unfortunately, this strategy was unsuccessful. The second strategy, which worked, involved performing a partial digestion, as one of the restriction enzyme sites was located within the NFAT insert itself. We then performed directional cloning into a new vector containing the Flag tag .