Difference between revisions of "Part:BBa K3941003"

 
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<partinfo>BBa_K3941003 short</partinfo>
 
<partinfo>BBa_K3941003 short</partinfo>
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<b><font size="+1">Summary</font></b>
  
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This part is originated from an endoglucanase (EG) gene that cleaves the internal beta-1,4-glucosidic bonds in cellulose. This part contains an amino acid mutation at 185 amino acid position.
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BBa_K3941003 is a codon-optimized (for <i>E. coli</i> DH5⍺) D185S mutated version of the endoglucanase (EG) gene that cleaves
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the internal beta-1,4-glycosidic bonds in cellulose. We optimized the sequence for expression and added a 6XHis at the
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end.
  
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K3941003 SequenceAndFeatures</partinfo>
 
  
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https://static.igem.org/mediawiki/parts/thumb/5/52/T--Saint_Joseph--Diagram-EGII-D185S.png/800px-T--Saint_Joseph--Diagram-EGII-D185S.png
  
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<font size="-2"><b>Figure 1:</b> Codon optimized EGII with a D185S mutation and a His-Tag</font>
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===Functional Parameters===
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<partinfo>BBa_K3941003 parameters</partinfo>
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<b><font size="+1">Introduction</font></b>
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EG2 is produced by <i>Trichoderma reesei</i> which is a fungi. Substrate specificity, binding properties, and cleavage products
 +
of EG2 were examined to evaluate its potential multiple enzymatic activities. Wild type EGII has a molecular weight of
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52 kDa and has an optimum pH of 5.0 and optimum temperature of 40°C and 50°C. EGII can maintain 89% of its endoglucanase
 +
activity at 40 °C and more than 80% at 50 °C for 60 min.
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<b><font size="+1">Design</font></b>
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https://2021.igem.org/wiki/images/thumb/6/61/T--Saint_Joseph--Diagram-EGII-D185S-Design.png/800px-T--Saint_Joseph--Diagram-EGII-D185S-Design.png
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<font size="-2"><b>Figure 2:</b> Design Process of EGII (D185S)</font>
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We mutated the EGII to use and later on optimized it to use in <i>E. coli</i> DH5⍺ bacteria. We also used <i>E. coli</i> BL21 to
 +
express our plasmids. To harvest our proteins, we added a His-tag (6XHis) at the end of our AA sequence. We changed the
 +
Aspartic acid from the 185th amino acid to Serine. 185th position is a functional hotspot and has a mutation score of 6
 +
on chain A.
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<b><font size="+1">Results</font></b>
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We have done a spectrophotometer absorbance analysis.
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https://2021.igem.org/wiki/images/7/77/T--Saint_Joseph--Nanodrop-EGII-D185S.png
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<font size="-2"><b>Figure 3:</b> The results of spectrophotometer absorbance analysis. The numerical columns are A230, A260, A280, A320, A260/A280, A260/A230 respectively</font>
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After that we have done an agarose gel electrophoresis.
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https://static.igem.org/mediawiki/parts/5/53/T--Saint_Joseph--Part-Agarose.png
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<font size="-2"><b>Figure 4:</b> The comparision between the backbone of the plasmid and EGII (D185S) is visible</font>

Revision as of 20:55, 17 October 2021


EGII (D185S) Summary

BBa_K3941003 is a codon-optimized (for E. coli DH5⍺) D185S mutated version of the endoglucanase (EG) gene that cleaves the internal beta-1,4-glycosidic bonds in cellulose. We optimized the sequence for expression and added a 6XHis at the end.


800px-T--Saint_Joseph--Diagram-EGII-D185S.png

Figure 1: Codon optimized EGII with a D185S mutation and a His-Tag



Introduction

EG2 is produced by Trichoderma reesei which is a fungi. Substrate specificity, binding properties, and cleavage products of EG2 were examined to evaluate its potential multiple enzymatic activities. Wild type EGII has a molecular weight of 52 kDa and has an optimum pH of 5.0 and optimum temperature of 40°C and 50°C. EGII can maintain 89% of its endoglucanase activity at 40 °C and more than 80% at 50 °C for 60 min.

Design


800px-T--Saint_Joseph--Diagram-EGII-D185S-Design.png Figure 2: Design Process of EGII (D185S)


We mutated the EGII to use and later on optimized it to use in E. coli DH5⍺ bacteria. We also used E. coli BL21 to express our plasmids. To harvest our proteins, we added a His-tag (6XHis) at the end of our AA sequence. We changed the Aspartic acid from the 185th amino acid to Serine. 185th position is a functional hotspot and has a mutation score of 6 on chain A.


Results

We have done a spectrophotometer absorbance analysis.


T--Saint_Joseph--Nanodrop-EGII-D185S.png

Figure 3: The results of spectrophotometer absorbance analysis. The numerical columns are A230, A260, A280, A320, A260/A280, A260/A230 respectively


After that we have done an agarose gel electrophoresis.


T--Saint_Joseph--Part-Agarose.png

Figure 4: The comparision between the backbone of the plasmid and EGII (D185S) is visible