Part:BBa_K3941001
EGII
Summary
BBa_K3941001 is a codon-optimized (for E.coli DH5âș) 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 isnât efficient for us, so we took out signal peptides. Parts such as CBM1 and linkers can augment the probability of protein folding and itâs 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 (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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