Materials and Methods

We grew E.Coli DH5a transformed with the plasmid C3 containing the immunotoxin with the strong promoter(BBa_K320006). As a negative control we used cultures transformed with the C3 plasmid alone. A culture volume of 100 ml was spinned down. The pellets as well as the supernatents were used as samples for a protein analysis. The pellets were resuspended in lysis buffer (prepared as described in the Knight protocol) containing urea and sonicated for 15 minutes. For the western blot the samples were run on an SDSpage gel and then transferred to a nitrocellulose membrane. The detection was accomplished using the following antibodies: Anti-his biotin as primary and Streptavidin-HPR as a secondary antibody. Additional to the primary antibody we applied Anti-his HRP. The supernatants were run through a filter device with a 5 kDa cutoff (“centricon”). These samples were also anayzed with a western blot as described above.