Part:BBa_K3941002
EGII (C99V)
BBa_K3941002 is a codon-optimized (for E. coli DH5âº) 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⺠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]
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