Part:BBa_K4202015
This part is a fusion protein of EutM and SpyCatcher , and it`s used for the bio-scafford
We get the information of the EutM from the BBa_K311004 and other paper. We want to construct a biology scaffold based on this protein and another system SpyCatcher-SpyTag system. Thus, we connect the Spycatcher to the C-terminal of the EutM via a GS linker. In order to enable the protein can be secreted out of the bacterium, we connected the SacB signal sequence of Bacillus subtilis to the N-terminal of EutM.SacB is a signal peptide used in the Sec-SRP (secretory signal recognition particle) pathway by B. subtilis. Signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached) through an appropriate secretory pathway Besides, a His tag is contained between the SacB and EutM for the purification, because the SacB signal sequence will be cleavaged after being secreted. This protein can be utilized with the HagT209C::SpyT588 to form the biological scaffold. This biological scaffold can be used for other area such as purification of sewage, enzyme reaction and so on.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Result:
We obtained the CDS and protein sequence of EutM, engineered SpyCatcher and SacB secretion signal sequence of Bacillus subtilis from NCBI database, and designed the fusion protein SP-EutM-spycatcher. To ensure that the designed fusion protein still has the ability to assemble, I-TASSER was used for homology modeling. The results showed that the recombinant protein could still form a reasonable spatial structure.
After EutM expression, we could clearly see that the culture showed a sticky character. That's what we expected.
We referenced the literature and finally determined the protein expression validation method through extensive experiments. Extracellular secretion and intracellular accumulation of scaffold building blocks by recombinant Bacillus strains was analyzed by preparing four different fractions from cultures for SDS-PAGE analysis.
We obtain the purified protein by His-tag Purification Resin. Concentrations of purified proteins were measured using the BCA Assay. For negative staining, 10 μL of protein was applied to the surface of a 200 μm formvar/carbon-coated copper grid . An equal volume of Trump’s fixative was added to the surface of the grid, and the protein/fixative drop was allowed to settle for 2 min. The surface of the grid was rinsed with 10 μL deionized water and excess fluid was removed. The protein on the grid was stained by applying 10 μL uranyl acetate (1%). Scaffolds were imaged on Phillips CM12 TEM with magnifications of x 20,000, x 50,000 and x 100,000.
The TEM image shows that when we overexpressed and purified EutM, the isolated protein readily precipitated out of solution as large crystalline arrays, with obvious hexameric organization and symmetry. These results indicated that EutM could serve as a building block for the design of a protein-based scaffolding system.
Then we tranformed the plasmid PHY-P43-HagT209C::SpyTag588-DT into Bacillus subtillis WB600 strain and inoculated into tetracycline resistant LB medium for overnight . Then we cultured the Bacillus subtillis WB600 containing PHY-HagT209C::SpyTag588(BBa_K4202006) and PHY-EutM-SpyCatcher in the tetracycline resistance SMM medium. After culturing 36h, we added the purified SpyCatcher-mRFP into the medium and cultured the bacteria for 12h. Then we utilized the confocal microscope(FLUOVIEW FV3000) to detect the cultures treated by SYTO .
We can clearly observe the green fluorescence at 507 nm and red fluorescence at 610nm. So we can conclude that the biological scafforld can be assembled reasonably.
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