Difference between revisions of "Part:BBa K320006:Experience"

Revision as of 21:38, 24 October 2010

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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.

User Reviews

UNIQc577b428c977681c-partinfo-00000000-QINU UNIQc577b428c977681c-partinfo-00000001-QINU

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 __NOTOC__
 This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 how you used this part and how it worked out.
-===Applications of BBa_K320006===
+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.
+[[Image:EPFL Gel1 western.jpg|thumb|right|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.  ]]
+[[Image:EPFL_Gels_2_western.jpg|thumb|buttom|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. ]]
 ===User Reviews===