Part:BBa_K3941003

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.

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

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

The mutation on the sequence made a lethal effect on the bacteria. We couldn't work on it anymore. But we could ran some analysis on it.

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 (D185S) is visible