Difference between revisions of "Part:BBa K1800000:Experience"

Revision as of 15:10, 27 September 2015

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The 2015 GSU iGEM team was able to successfully express and characterize the mambalgin protein in E. coli. After inducing with IPTG overnight, the cell cultures were centrifuged and disrupted with a French press. Mambalgin was isolated using affinity chromatography, utilizing the 6x His tag in the construct.

A coomassie stain was performed to visualize the proteins. A band indicating a protein of ~9 kda – the size of the mambalgin for E. coli construct – was seen in the eluate fraction which is indicative of our desired protein. However, this band may also contain any native proteins that migrate below 11 kD (final elution contained the expected product). However the flowthrough and 2 washes did not have product, meaning the mambalgin protein was bound to the NiTA resin.

MAMBA.E.coli_Western_blot.THISONE.jpg
In addtition, an anti-myc Western was performed to confirm the presence of mambalgin and a signal was observed at the expected size. An additional band at ~35 kd was detected however. We believe that this may be due to aggregates formed by non-specific disulfide bond formation.

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UNIQ9b73cc647cabb4e6-partinfo-00000000-QINU UNIQ9b73cc647cabb4e6-partinfo-00000001-QINU

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-A coomassie stain was performed to visualize the proteins. A band indicating a protein of ~9 kda – the size of the mambalgin for E. coli construct – was seen in the eluate fraction which is indicative of our desired protein. However, this band may also contain any native proteins that migrate below 11 kD. <br>
+A coomassie stain was performed to visualize the proteins. A band indicating a protein of ~9 kda – the size of the mambalgin for E. coli construct – was seen in the eluate fraction which is indicative of our desired protein. However, this band may also contain any native proteins that migrate below 11 kD (final elution contained the expected product). However the flowthrough and 2 washes did not have product, meaning the mambalgin protein was bound to the NiTA resin.  <br>
 https://static.igem.org/mediawiki/2015/d/dd/MAMBA.E.coli_Western_blot.THISONE.jpg <br>
-An anti-myc Western was performed to confirm the presence of mambalgin and a signal was observed at the expected size. An additional band at ~35 kd was detected however. We believe that this may be due to aggregates formed by non-specific disulfide bond formation.
+In addtition, an anti-myc Western was performed to confirm the presence of mambalgin and a signal was observed at the expected size. An additional band at ~35 kd was detected however. We believe that this may be due to aggregates formed by non-specific disulfide bond formation.
 ===Applications of BBa_K1800000===