Difference between revisions of "Part:BBa K3941002"
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<partinfo>BBa_K3941002 short</partinfo> | <partinfo>BBa_K3941002 short</partinfo> | ||
− | + | BBa_K3941002 is a codon-optimized (for E. coli DH5⍺) C99V 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. | |
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+ | https://static.igem.org/mediawiki/parts/thumb/0/01/T--Saint_Joseph--Diagram-EGII-C99V.png/800px-T--Saint_Joseph--Diagram-EGII-C99V.png | ||
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+ | <font size="-2"><b>Figure 1:</b> Codon optimized EGII with a C99V mutation and a His-Tag</font> | ||
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+ | <b><font size="+1">Introduction</font></b> | ||
+ | EGII is produced by <i>Trichoderma reesei</i> which is a fungi. Substrate specificity, binding properties, and cleavage products of EGII were examined to evaluate its potential multiple enzymatic activities. | ||
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+ | <b><font size="+1">Design</font></b> | ||
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+ | https://static.igem.org/mediawiki/parts/thumb/5/58/T--Saint_Joseph--Diagram-EGII-C99V-Design.png/799px-T--Saint_Joseph--Diagram-EGII-C99V-Design.png | ||
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+ | <font size="-2"><b>Figure 2:</b> Schema of how did we designed our EGII (C99V)</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 Cystine from the 99th amino acid to Valine. | ||
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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://static.igem.org/mediawiki/parts/0/00/T--Saint_Joseph--Nanodrop-EGII-C99V.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 (C99V) is visible</font> | ||
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<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here |
Revision as of 18:51, 14 October 2021
EGII (C99V)
BBa_K3941002 is a codon-optimized (for E. coli DH5⍺) C99V 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.
Figure 1: Codon optimized EGII with a C99V mutation and a His-Tag
Introduction EGII is produced by Trichoderma reesei which is a fungi. Substrate specificity, binding properties, and cleavage products of EGII were examined to evaluate its potential multiple enzymatic activities.
Design
Figure 2: Schema of how did we designed our EGII (C99V)
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 Cystine from the 99th amino acid to Valine.
Results
We have done a spectrophotometer absorbance analysis.
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.
Figure 4: The comparision between the backbone of the plasmid and EGII (C99V) is visible
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]