Part:BBa_K2155005
Contents
CBM3b---Components of interacting proteins
Usage and Biology
This domain is part of the Cellulosome Integrating Protein A (CipA), which forms part of the cellulosome of Clostridium thermocellum. The cellulosome is an extracellular protein scaffold attached to the bacterial cell wall and displays cellulose-degrading enzymes (cellulases). Cellulose Binding Domain (CBD) named CBM3b has high-affinity on low-cost solid regenerated amorphous cellulose (RAC). Besides, this domain is also thought to increase stability of the secreted scaffold. With CBM3b, we could purify the our scaffold-enzyme complex easily by specially binding with RAC.
Improvement
This year, we reduced 27bp of a registered part(BBa_K1830003, 12bp at 5’ end and 15bp at 3’ end) and then created our new part CBM3b. According to a paper published by Stéphanie Perret in 2013, Both modules(CBM3a and CBM3b) bind to phosphoric acid swollen cellulose (PASC) with the same affinity. So we use a smaller module(CBM3b) to achieve the same function in our project.
Contribution From UESTC-China 2021
Group: iGEM Team UESTC-China 2021
Author: Liyan Wu
Characterization from UESTC-China 2021
The CBM3 domain can highly bind with cellulose, which we need much on our paper deinking project as paper is rich in cellulose, and we connected different original cohesin domains at two sides to bind with dockerin domain linked to cellulase or xylanase. These enzymes can work together to deink the ink on the paper. We use pichia pastoris to express these enzymes and cellulosome components, so we did codon optimized for yeast expression and our part number is BBa_K3819004.
- Figure 1. Gene squence alignment of CBM3 from iGEM16_NWPU and UESTC-China
- Figure 2. Amino acid squence alignment of CBM3 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 CBM3 domain, as a myc tag has been linked, we can observe its expression as the following results of Dot-Blot analysis.
- Figure 3. The Dot-Blot results of recombined GS115(pScaffold) fermentation broth. The sample was gained after fermentation in YPD for 96h. Scaffold(Myc-Tag) was tested by adding 2ul fermentation supernatant on NC membrane and exposing 60s.
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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