Difference between revisions of "Part:BBa K3941001"

Revision as of 11:01, 19 October 2021

EGII

Summary

BBa_K3941001 is a codon-optimized (for E.coli DH5⍺) 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 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 design of the EGII

262 to 590 and 765 to 1692 nucleotide sequences from Trichoderma reesei were used in the project since the EGII gene is too massive to work on. Signal peptides were removed since enzyme release is unnecessary for the project. Parts such as CBM1 and linkers were removed because of the augmentation of protein folding probability which is a complicated process for a prokaryotic organism. Histidine tag was added at the end of the AA sequence which made protein purification easier.

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 is visible

Lastly we conducted a CMCase Activity Analysis. EGII has an absorbance of 0,054 and enzyme activity of 2.1e-2 umol/L.min.

Figure 5: Calibration Curve for CMCase Activity Analysis

Figure 6: Graphs of EGII's CMCase Activity Analysis

References

- Tjandra, Kezia & Sari Dewi, Kartika & Fuad, Asrul Muhamad & Anindyawati, Trisanti. (2020). Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter. Indonesian Journal of Biotechnology. 25. 127. 10.22146/ijbiotech.55604.

- Akbarzadeh, Ali & Pourzardosht, Navid & Dehnavi, Ehsan & Siadat, Seyed & Zamani, Mohammadreza & Motallebi, Mostafa & Jamnani, Farnaz & Aghaeepoor, Mojtaba & Barshan-tashnizi, Mohammad. (2018). Disulfide bonds elimination of endoglucanase II from Trichoderma reesei by site-directed mutagenesis to improve enzyme activity and thermal stability: An experimental and theoretical approach. International Journal of Biological Macromolecules. 120. 10.1016/j.ijbiomac.2018.09.164.

Sequence and Features