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 0.2 mg purified SpyCatcher-mRFP per 5 ml medium into the medium and cultured the bacteria for 12h.Besides, we settled WB600 group and calcium carbonate group for control. Then we utilized the confocal laser scanning 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, while the group for control showed negative outcome. So we could conclude that the biological scafforld can be assembled reasonably.
Then in order to test the feasibility of the combination between biological scaffold and calcium carbonate precipitation as a whole, we designed a co-culture experiment. The Bacillus subtilis with CA(BBa_K4202004), EutM, Hag-SpyTag588(BBa_K4202006) were cultured in 50 ml LB medium at 25 oC, 220 rpm, pH 8.0 for 4 days. At the first day and the third day 500μl 1M CaCl2 was added into the medium. Besides, a group without the Bacillus subtilis containing CA was settled for control.
After the induction, we utilized the microscope to preliminary detect the cultures. Then we utilized the fluorescence microscope to detect the cultures treated with SYTO dye to verify that there are bacteria in the briquette of the cultures.
Then we managed to test the yield of the complex. We sampled 800μl cultures into the 1.5 ml ep tube and the method showed below was adapted for the determination of the yield of the whole complex and the calcium carbonate precipitation respectively.
Step 1: The EP tubes were weighed before separating the bacteria solution to obtain M1 and we sampled 1ml cultural solution into the 1.5 ml ep tube.
Step 2: We collected the complex at 12000 rpm 2min. The surnatant was discarded, fully dried and weighed to obtain M2.
Step 3: We used a high concentration of hydrochloric acid to dissolve the precipitate. After there were no obvious bubbles, we utilized the pH dipstick to test and confirm that the pH was less than 7.
Step 4: After waiting for full reaction, we added triploid volume of absolute ethanol. Then， we collected the precipitation at 12000rpm for 2min, discarded the supernatant, dried it thoroughly and weighed it to obtain M3.
Step 5: Calculate the yield according to the following formula.
A1=(M2-M1 ) / V * 100%
A2=(M3-M2) / V * 100%
Result: The results showed a high variation rate, but we could still conclude that the precipitation was a mixture of bacteria and mineralized products.
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