Difference between revisions of "Team:Dalhousie/Improve"

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This part is the coding region for an endoglucanse from Ruminiclostridium thermocellum that was previously created by the iGEM16_Dalhousie_NS_Halifax  
 
This part is the coding region for an endoglucanse from Ruminiclostridium thermocellum that was previously created by the iGEM16_Dalhousie_NS_Halifax  
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(<a href="http://parts.igem.org/Part:BBa_K2160000" style="color: #C1D35D">BBa_K2160000</a>). Its function is to cleave internal Beta-1,4-D-glycosidic bonds in the cellulose crystal to release the disaccharide cellobiose.</br></br>
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(<a href="http://parts.igem.org/Part:BBa_K2160000" style="color: #C1D35D">BBa_K2160000</a>). Its function is to cleave internal beta-1,4-D-glycosidic bonds in crystalline cellulose to release the disaccharide cellobiose.</br></br>
  
 
<font color= "#C1D35D">Improvement</font></br>
 
<font color= "#C1D35D">Improvement</font></br>
  
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<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 <i>E. coli</i> to digest cellulose.</font></br>
+
<font color= "#ffffff">We improved the endoglucanase part by adding a C-terminal His-tag and an 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 <i>E. coli</i> to digest cellulose.</font></br>
  
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<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>
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<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>
  
 
<div style="background-color: rgba(61,85,28, 0.5); width: 70%; height: 70%; padding-left: 50px; padding-right: 50px; float: center; margin-top: 20px; margin-left: 70px; margin:auto;">
 
<div style="background-color: rgba(61,85,28, 0.5); width: 70%; height: 70%; padding-left: 50px; padding-right: 50px; float: center; margin-top: 20px; margin-left: 70px; margin:auto;">

Revision as of 00:06, 1 November 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 crystalline cellulose to release the disaccharide cellobiose.

Improvement
We improved the endoglucanase part by adding a C-terminal His-tag and an 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.
Figure 1. Western blot probing for 6xHis Tag


Part Number
Our part can be found here: BBa_K2160000

References

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.