Difference between revisions of "Part:BBa K3098017"

Latest revision as of 15:51, 21 October 2019

Strep-dCBM3a

This part is composed of the 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 for the bacterial cellulose than CBM3a. And the monomeric strepvidin domain at N-terminus can bind to biotinylated compounds. The part can act as a adaptor which makes the binding of biotinylated functional compounds and the bacterial cellulose possible.

Usage and Biology

The part is composed of the dCBM3a domain and the monomeric streptavidin domain.The dCBM3a domain is the dimer of the CBM3a with three linkers peptide 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. Sequence and Features

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 dCBM3a, CBM3a and the negative control sfGFP. Then the samples will be mixed with bacteria cellulose homogenate. The fluorescence residues were measured after incubation 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 the supernatent after incubation has obviously lower amount of the dCBM3a proteins, while there were a lot of dCBM3a bound to the biotinylated magnetic beads.