Difference between revisions of "Team:Dalhousie/Improve"
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| − | <img src= "https://static.igem.org/mediawiki/2017/ | + | <img src= "https://static.igem.org/mediawiki/2017/3/3e/Dalbackt.jpg" style="top:0px; left:0px; padding-bottom:0px; position:fixed; width:100%; height: 100%; z-index:-100; " align="center" height="70%" width="70%"> |
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<font color= "#C1D35D">Background</font></br> | <font color= "#C1D35D">Background</font></br> | ||
| − | + | This part is the coding region for an endoglucanse from Ruminiclostridium thermocellum that was previously created by the iGEM16_Dalhousie_NS_Halifax | |
| − | (<a href="http://parts.igem.org/Part:BBa_K2160000" style="color: #C1D35D">BBa_K2160000</a>). Its function is to cleave internal | + | (<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> | ||
| − | <font color= "#ffffff">We improved the endoglucanase part by adding a C-terminal His-tag and | + | <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> |
| − | <font color= "#ffffff">Using a western blot to probe for the | + | <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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| + | <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;"> | ||
| + | <center><img src="https://static.igem.org/mediawiki/2017/2/2d/Endoglucoptimized.png" height="45%" width="45%"></center> | ||
| + | </div> | ||
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<center><font color= "#ffffff"> Figure 1. Western blot probing for 6xHis Tag</Center></font></br></br> | <center><font color= "#ffffff"> Figure 1. Western blot probing for 6xHis Tag</Center></font></br></br> | ||
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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></br> |
<font color= "#C1D35D">References</font></br> | <font color= "#C1D35D">References</font></br> | ||
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<div class="inner"> | <div class="inner"> | ||
<p style="text-align: center; "> | <p style="text-align: center; "> | ||
| − | <img src="https://static.igem.org/mediawiki/2017/8/ | + | <a href="http://www.plosibilities.wordpress.com"><img src="https://static.igem.org/mediawiki/2017/archive/8/8c/20171031235427%21Dalscreen.png" height="20%" width="20%" ></a> |
<img src="https://static.igem.org/mediawiki/parts/d/d7/Porcupinelogo2017.png" height="20%" width="20%" > | <img src="https://static.igem.org/mediawiki/parts/d/d7/Porcupinelogo2017.png" height="20%" width="20%" > | ||
<img src="https://static.igem.org/mediawiki/2017/e/ef/Whitetigerlogo.png" height="20%" width="20%" > | <img src="https://static.igem.org/mediawiki/2017/e/ef/Whitetigerlogo.png" height="20%" width="20%" > | ||
Latest revision as of 00:54, 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.
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.
