Part:BBa_K320006:Experience

A: The Immunotoxin is expressed and appears in the supernatant

Growth curve of E. coli expressing the Immunotoxin: In blue, we can see the growth curve of E.coli containing the empty C3 plasmid. In red, we can see the growth of E.coli transformed with the C3 plasmid containing the Immunotoxin sequence. We could conclude that the expression of the Immunotoxin in high concetrations may prevent E.coli from growing. The spikes could indicate the clumping of cells due to Immunotoxin's action.

We tested expression of the immunotoxin in E.Coli (see [http://2010.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Project/Materials_Methods Materials and Methods] for details). In a western blot analysis of whole cell lysates we could see bands corresponding to full length immunotoxin and possibly degraded fragments of the protein (see figure). The immunotoxin contains a PelB sequence that targets it for secretion into the periplasm. We concentrated the supernatant of both the immunotoxin and a control culture by running it through a filtering device with a 5 kDa cut-off. Running a western blot with these samples (see figure) we verified that the immunotoxin was found in the supernatant as expected.

Gel1 (Lysates): We can see distinctive molecular fragments in the immunotoxin lane: The upper fragments (marked with red and purple arrows) match the molecular size of the immunotoxin of about 39 kDa. For the shorter of the two fragments the export tag pelB may have been cut off. The smaller sized fragment (green arrow) of about 20 kDa is most probably part of the degraded immunotoxin. Gel2 (Supernatants): A fragment of the correct size is detected in the supernatant. The smaller fragments (green and yellow arrows) probably correspond to smaller parts of the degraded immunotoxin.

We tested expression of the immunotoxin in E.Coli (see [http://2010.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Project/Materials_Methods Materials and Methods] for details). In a western blot analysis of whole cell lysates we could see bands corresponding to full length immunotoxin and possibly degraded fragments of the protein (see figure). The immunotoxin contains a PelB sequence that targets it for secretion into the periplasm. We concentrated the supernatant of both the immunotoxin and a control culture by running it through a filtering device with a 5 kDa cut-off. Running a western blot with these samples (see figure) we concluded that the immunotoxin was secreted as expected.

We can see distinctive molecular fragments in the immunotoxin lane which are not present in the negative control. The upper fragments (marked with red and purple arrows) seem to match the molecular size of the immunotoxin of about 39 kDa. For the shorter of the two fragments the export tag pelB may have been cut off. The smaller sized fragment of about 20 kDa in the whole cell lysate is most probably part of the degraded immunotoxin.

The band in the positive control (BH2, kDa) shows that the western blot worked. We see a clear band of the correct site (red arrow) which proves that the exportation works as expected and the immunotoxin is actually secreted. Obviously there is no need for a lysis device.

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