Difference between revisions of "Part:BBa K2467000"

Latest revision as of 05:52, 21 October 2021

DsbA DspB emGFP

To improve part BBa_K1659211, DsbADspB (dispersin exported to the extracellular using the DsbA signal sequence (KKIWLALAGLVLAFSASA)), we fused it to emGFP in order to measure secreted protein using fluorescence. We amplified the DsbADspB fragment and incorporated EcoR1 and BamH1 restriction sites. The gene was ligated onto the multiple cloning site on the pRSET emGFP vector which resulted in fluorescently tagged DspADspB. This fragment will then be moved to pSB1C3. This allows for easy fluorescence detection in the supernatant contributed by the exported DspADspB. Projects dealing with disruption of Biofilms can easily follow the release of the dispersin enzyme into the extracellular environment to degrade the biofilm polysaccharides

BJ101ID 2021's Contribution

Contribution from BJ101ID-iGEM2021：

[1] Liu, Z. , Zhao, Z. , Zeng, K. , Xia, Y. , & Xie, H. . (2021). Magnetic Immobilization of Dispersin B with Activity in Degradation of Bacterial Biofilm.

Team CMUQ of 2017 used the DspB gene in their project. Our team had found a new research done by the Wuhan University of Technology in March 2021 that also relates to the DspB. The research name was Magnetic Immobilization of Dispersin B with Activity in Degradation of Bacterial Biofilm. These two research both approached DspB function in the same way but used them differently, as we were able to see how one segment of genes could be used to complete so many different works. In this section, I will explain how a previous gene used by an iGEM team was modified an applicated into a different research Biofilms are formed by a extracellular polymeric matrix, composed of polysaccharide, lipids and nucleic acids, that surrounds a group of bacteria. They are functioned to reduce the sensibility of the bacteria toward antibiotics. Biofilms are involved in over 65% if bacterial infection and could lead to very serious consequences when not responded correctly. Disperisin B (DspB), from the glycoside hydrolase family, is a catalyst to the degradation of biofilms. When a bacterial cell is released to the external environment to build new biofilms, DspB are functioned to degrade the biofilm. Previous methods are usually through chemical and enzyme bonding. The new approach used a physical method to make the degradation process more stable and controllable. Magnetoreceptor (MagR), a fusion partner for the functional immobilization of proteins on a magnetic surface, was inserted to the C-terminus of DspB to form a recombinant protein DspB-MagR. This protein will be then purified by the Ni-NTA affinity chromatography and immobilized on the Fe3O4@SiO2 nanoparticles. The homogeneity of the protein after purification and immobilization was 95%, meaning that theoretically the loading process did not alter the protein greatly. The researchers carried out bioactivity tests on the loaded protein and unloaded protein to see the difference. For pH sensitivity, there wasn’t a big change after loading as the highest sensitivity is still at 6. For temperature, the highest activity state raised from 30°C to 37°C after loading. This supports that the loaded protein will be more suited for medical purposes since its highest activity state is at a temperature similar to the regular body temperature. It was also tested that through a long duration, the loaded protein still kept a higher activity state than the unloaded protein.

The degradation function of DspB-MagR, immobilized DspB-MagR and Fe3O4@SiO2 was tested on the biofilm of staphylococcus aureus. Fe3O4@SiO2 showed no effect to detaching bacterial biofilms, DspB-MagR was able to remove 10% while the loaded DspB-MagR was able to remove 50%. The immobilized protein was also tested for biofilms of other bacterium like the Bacillus Cereus and Staphylococcus sp and showed significant removal rate. These results proved that using Fe3O4@SiO2 to immobilize the DspB-MagR recombinant protein is an alternative method for biofilm removal.

Figure 1. Expression of recombinant DspB-MagR.

Figure 2. Immobilization of DspB-MagR and characterization of Fe3O4@SiO2 nanoparticles with loaded DspBMagR.

Figure 3. Sensitivity of DspB-MagR to temperature, pH, storage time before and after loading on Fe3O4@SiO2 nanoparticles.

Figure 4. Bioactivity of immobilized DspB-MagR on detaching bacterial biofilm.

Sequence and Features