Difference between revisions of "Team:Dalhousie/Improve"

Line 208: Line 208:
  
 
<font color= "#ffffff">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.</font></br>
 
<font color= "#ffffff">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.</font></br>
 +
 +
<font color= "#ffffff">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. </font></br>
 +
 +
<img src"https://static.igem.org/mediawiki/2017/2/2d/Endoglucoptimized.png"/>
 
http://parts.igem.org/Part:BBa_K2331000      LINKKK
 
http://parts.igem.org/Part:BBa_K2331000      LINKKK
  

Revision as of 00:48, 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.
http://parts.igem.org/Part:BBa_K2331000 LINKKK

Learn more... hopefully have links to next pages here