Difference between revisions of "Team:Dalhousie/Improve"
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<font color= "#C1D35D">Part Number</font></br> | <font color= "#C1D35D">Part Number</font></br> | ||
| − | <font color= "#ffffff">Our part can be found here: <a href="http://parts.igem.org/Part:BBa_K2331004" style="color: #C1D35D">BBa_K2160000</a></br></br> | + | <font color= "#ffffff">Our part can be found here: <a href="http://parts.igem.org/Part:BBa_K2331004" style="color: #C1D35D">BBa_K2160000</a></br> |
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| + | <font color= "#C1D35D">References</font></br> | ||
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| + | <font color= "#ffffff">Sockolosky, J. and Szoka, F. (2013). Periplasmic production via the pET expression system of soluble, bioactive human growth hormone. Protein Expression and Purification, 87(2), pp.129-135.</font> | ||
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Revision as of 01:15, 27 October 2017
Improve
Part Improvement
Background This part is the coding region for an endoglucanse from Ruminiclostridium thermocellum that was previously created by the iGEM16_Dalhousie_NS_Halifax (BBa_K2160000). Its function is to cleave internal Beta-1,4-D-glycosidic bonds in the cellulose crystal to release the disaccharide cellobiose. Improvement We improved the endoglucanase part by adding a C-terminal His-tag and a N-terminal PelB sequence. The C-terminal His-tag allows identification via western blot or immuno-fluorescence, and protein purification. The PelB sequence is a localization sequence that traffics the protein to the periplasm (Sockolosky & Szoka, 2013). This is especially important for our project because we need to get all the enzymes out of the E. coli to digest cellulose. Using a western blot to probe for the HIS-Tag, we were able to show expression of our optimized endoglucanase. The main species traveled at 46 kDa, which was the predicted migration of endoglucanase with a HIS-tag and PelB sequence. A secondary, smaller species was seen at ~30 kDa. The 30 kDa species can be explained due to a second methionine codon with an imperfect ribosomal binding sequence 5-10 bp upstream from the Met codon.
Sockolosky, J. and Szoka, F. (2013). Periplasmic production via the pET expression system of soluble, bioactive human growth hormone. Protein Expression and Purification, 87(2), pp.129-135.
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