Usage and Biology
It is from endoglucanase EngE of C.cellulovorans, and have the property to assemble automatically with a cohesin module from CbpA. Endoglucanase E (EngE) is one of the three major subunits of the Clostridium cellulovorans cellulosome. EngE plays an anchoring role for cellulosomes through the interaction of its dockerin domain with a CbpA cohesin. In our project, we fused TalB with CcsDock, and then CcsDock could bind with CcsCohesin1 which is part of our scaffold protein. Therefore, our core enzyme could form a enzyme complex, and the catalytic efficiency could be greatly improved.
Contribution From UESTC-China 2021
Group: iGEM Team UESTC-China 2021
Author: Liyan Wu
Characterization from UESTC-China 2021
We use this dockerin domain from C.cellulovorans to connected with cellulase by two (GGGGS)3 linkers, and it can bind with CcsCohesin which is part of our scaffold protein, and ultimately anchor our cellulase and xylanase to the cell surface of Pichia pastoris to improve efficiency of deink. Because we chose Pichia pastoris as the chassis organism, we have done codon optimized for yeast expression, and our part number is BBa_K3819066.
- Figure 1. Gene squence alignment of CcsDockerin from iGEM16_NWPU and UESTC-China
- Figure 2. Amino acid squence alignment of CcsDockerin from iGEM16_NWPU and UESTC-China
The two figures above show that the amino acid sequence of the two parts are the same but the gene sequence is not completely the same.
We finally successfully express the dockerin domain, as a 6×His tag has been linked, we can observe its expression as the following results of Dot-Blot analysis and enzyme activity.
- Figure 3. The Dot-Blot results of recombined GS115(pEG1) fermentation broth. The sample was gained after fermentation in YPD for 96h. EG1(His-Tag) was tested by adding 2ul fermentation supernatant on NC membrane and exposing 60s.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC