Part:BBa_K2668000

Cerberus tetrameric (E. coli)

This part consists of a CBM3a fused with a Streptavidin on its N terminus endogenous linker and containgin a UAG codon on its C terminus linker for amber suppression unnatural amino acid incorporation.

Improvement: WHU-China 2019

Group: WHU_China iGEM 2019
Author: Junfan Chen
WHU-China 2019 submits a new part called dCBM3a which is a improved dimer of the CBM3a ( form BBa_K2668000 ) fused another monomer at the C-terminus and the monomeric streptavidin at the N-terminus. The dCBM3a domain has a higher affinity to the bacterial cellulose than CBM3a. And the monomeric strepvidin domain at N-terminus can bind to biotinylated compounds. The part can act as an adaptor which makes the binding of biotinylated functional compounds and the bacterial cellulose possible.

The part is designed to compose two of the CBM3a domain and the monomeric streptavidin domain.The dCBM3a domain is the dimer of the CBM3a with three linker peptides between the two CBM3as, at the N-terminus and the C-terminus of the domain. The linker region between two CBM3as contains 37 amino acids. The first five amino acids are from the N-endogenous linker, and the rest is from the C-endogenous linker. The N-terminus linker of the dCBM3a domain is the N-endogenous linker of the CBM3a. And the C-terminus of it is the same as the C-endogenous linker of the CBM3a.

Result 1.Functional Detection of Cellulose Binding Domain
SDS-PAGE detection by using of the lysate of the engineered bacteria showed obvious target bands, among which the molecular weight of CBM3a with streptavidin was about 48kDa and that of dCBM3a with sterptavidin was about 89kDa.

We set three groups in this experiments including dCBM3a, CBM3a and the negative control sfGFP. Then the samples will be mixed with bacteria cellulose homogenate. The fluorescence residues were measured after the incubation of the bacterial lysate with BC membrane and washing of them. We found that the dCBM3a that we improved has higher affinity with bacteria cellulose than CBM3a.

2.Functional detection of Streptomycin Domain
Streptavidin can specifically bind to biotin, so biotinylated magnetic beads were used to detect the streptomycin domain of dCBM3a. After incubating the biotinylated magnetic beads with engineered bacteria lysate and washing, it was found that the target protein was successfully attached to the biotinylated magnetic beads by SDS-PAGE detection. We can see that in the supernatent after incubation, there was obviously lower amount of the dCBM3a proteins, while there were a lot of dCBM3as bound to the biotinylated magnetic beads.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 376
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 324