Part:BBa_K2155012

Synthetic Protein Scaffoldin

Usage and Biology

The protein scaffoldin is an assembly platform for ligand coupled target enzymes (figure 1). It was designed by the Keasling Lab in 2009 in order to improve the yield and production rate of metabolic processes. The association of target enzymes with the scaffoldin mimic naturally occurring catalysation cascades. Then reaction efficiencies are optimized through the passing on of intermediates between co-located enzymes. The quick processing of intermediates can help to overcome negative production effects including unstable or toxic intermediates, metabolic bottlenecks or accumulation of undesired intermediates (Dueber et al. 2009). The protein scaffoldin consists of three different protein binding domains namely the Ct cohesion (BBa_K2155006), Ccs cohesion1 (BBa_K2155007) and Ccs cohesin2 (BBa_K2155008) domains. The coding sequence has been revised in E.coli for the usage as a BioBrick.

Contribution From UESTC-China 2021

Group: iGEM Team UESTC-China 2021

Author: Liyan Wu

Characterization from UESTC-China 2021

Scaffoldin is the skeletal protein of cellulosome. BBa_K2155012 put the CBM domain on the end and the remaining cohesin domains are on one side of the CBM. We put cohesin domain on both sides of CBM domain, and use rigid linker (Pro/ THR-rich, BBa_K3819025), these improvements reduced the steric hindrance of adjacent domain proteins, thus reducing the impact on their function and enabling them to function normally. In addition, we added a dockerin domain on the end to bind with the cohesin domain linking to SdbA to anchor to the surface of Pichia pastoris cells.

Futhermore, this year our chassis organism is Pichia pastoris GS115, so we have done codon optimization for yeast expression, and the sequence alignment result of scaffoldin gene from team iGEM16_NWPU and our team are as Figure1. In addition, the amino acid sequence alignment result of cellulosomal-scaffolding protein A [Acetivibrio thermocellus] and our self-design scaffoldin protein are shown as Figure2.

Figure 1. Gene squence alignment of Scaffoldin from iGEM16_NWPU and UESTC-China.

Figure 2. Amino acid sequence alignment of cellulosomal-scaffolding protein A [Acetivibrio thermocellus] and our self-design scaffoldin protein

We have also successfully expressed our scaffoldin (BBa_K3819012), which means the sequences after codon optimization can be well expressed in yeast cells. Shown as the following results of Dot-Blot analysis.

Figure 3. The Dot-Blot results of recombined GS115(pScaf) fermentation broth. The sample was gained after fermentation in YPD for 96h. Scaffoldin(Myc-Tag) was tested by adding 2ul fermentation supernatant on NC membrane and exposing 60s.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]