Team:WHU-China/Improve

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Improve

Existing Part:BBa_K2668000

New Part:BBa_K3098017

WHU-China 2019 submit a new part called dCBM3a which is an improved dimer composed of CBM3a fused with another CBM3a monomer at C-terminus and the streptavidin monomer at the N-terminus. The dCBM3a domain has a higher affinity for the bacterial cellulose than CBM3a. And the monomeric streptavidin domain at N-terminus can bind to biotinylated compounds. The part can act as an adaptor which makes the binding of biotinylated functional compounds with the bacterial cellulose possible.

The part is designed to compose two of the CBM3a domain and a monomeric streptavidin domain. dCBM3a domain is the dimer of the CBM3a with three linker peptides, which are located in N-terminus and C-terminus of dCBM3a domain and between the two CBM3a domains. The linker between two CBM3a domains contain 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 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 streptavidinwas about 89kDa.

We set three groups in this experiment: dCBM3a, CBM3a and the negative control--sfGFP. Then the samples will be mixed with bacteria cellulose homogenate.

The fluorescent residues were measured after incubating the bacterial lysate with BC membrane (pre-dried in a black 96-well plate) and washing for a few times. The higher the fluorescence intensity of the residues, the higher affinity of the CBD with bacterial cellulose is. According to the results below, we found that dCBM3a we improved has higher affinity with bacteria cellulose than CBM3a.

2.Functional detection of Streptavidin Domain

Streptavidin can specifically bind to biotin, so we decided to use biotinylated magnetic beads to detect the existence of streptavidin domain of dCBM3a. After incubating the biotin magnetic beads with engineered bacteria lysate and washing, it was found that the target protein was successfully attached to the biotin magnetic beads by SDS-PAGE detection. We can see that the supernatant after incubation has obviously lower amount of the dCBM3a proteins, while there were a lot of dCBM3a bound to the biotin magnetic beads.