Difference between revisions of "Team:Dalhousie/Improve"

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<p>This page is used by the judges to evaluate your team for the <a href="https://2017.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2017.igem.org/Judging/Awards"> award listed above</a>. </p>
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<h1>Improve</h1>
 
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<p>For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Regisrty. Please include a link to your improved part on this page.</p>
 
  
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<h3>Gold Medal Criterion #2</h3>
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<p><b>Standard Tracks:</b> Improve the function of an existing BioBrick Part. The original part must NOT be from your 2017 part number range. If you change the original part sequence, you must submit a new part. In addition, both the new and original part pages must reference each other. This working part must be different from the part documented in bronze #4 and silver #1.
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<b>Special Tracks:</b> Improve the function of an existing iGEM project (that your current team did not originally create) and display your achievement on your wiki.</p>
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<div class="top" ><div class="title" >Improve</div></div>
  
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<h1><font color= "#C1D35D">Part Improvement</font></h1></br>
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<font color= "#C1D35D">Background</font></br>
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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
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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>
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<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 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 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;">
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<center><img src="https://static.igem.org/mediawiki/2017/2/2d/Endoglucoptimized.png"  height="45%" width="45%"></center>
 
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<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>
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<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>
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<font color= "#C1D35D">References</font></br>
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<p class="hangingindent"><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> </p>
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<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>
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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.
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.