Difference between revisions of "Part:BBa K3941001"

Revision as of 18:50, 14 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 how did we created our EGII

We separated 262 to 590 and 765 to 1692 nucleotide sequences from Trichoderma reesei to use as our genomes are too massive. Signal peptides cause bacteria to release enzymes but for our project it isn’t efficient for us, so we took out signal peptides. Parts such as CBM1 and linkers can augment the probability of protein folding and it’s a complicated process for a prokaryotic organism. We took them out too. We added histidine tags to our enzymes. Histidine tags can bind to nickel, and with a nickel solution we can isolate proteins easily.

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

Sequence and Features