Difference between revisions of "Part:BBa K3941002"

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