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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE 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.

Fig 1The result of I-TASSER

After EutM expression, we could clearly see that the culture showed a sticky character. That's what we expected.

Fig 3 Observed EutM secretion property

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.

Fig 4 Experimental workflow used for the analysis of EutM scaffold building block secretion and expression by engineered B. subtilis.

Fig 5 SDS-PAGE and Coomassie brilliantblue staining results of EutM-SpyCatcher protein

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.

Fig 6 Protein-based scaffolding system. Negative stain TEM of the white precipitate formed in the tube shows crystalline EutM scaffolds composed of hexameric tilesthat are assembled from structurally ordered arrays of EutM hexamers (red line indicates ordered crystalline lattice). Insert highlights individual EutM hexamers.

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.

Fig 7The result of fluorescence confocal microscope

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.

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 dyed to verify that there are bacteria in the briquette of the cultures. We also settled a control group which contained only calcium carbonate.

Fig 8 A: The culture medium after natural settlement for 10min.The yellow sediment and small partials of calcium carbonate can be clearly observed. B: The cultures were observed at 400* microscope. The small entity indicated by the arrow might be the complex of biological scaffold and calcium carbonate.

Fig 9 The image of the confocal microscope(FLUOVIEW FV3000). The bacterium were stained to indicate.The arrow noted the sediment.

Result: We observed that in the coculture group, the sediment possessed green fluorescent, while the control group did not. So we concluded that the sediment was the complex of bacterium and calcium carbonate.

Step 1: The EP tubes were weighed(M1) before separating the bacteria solution to obtain M1 and we sampled 0.8ml cultural solution into the 1.5 ml ep tube. And we set five parallel experiments and numbered them 1-5.

Step 2: We collected the complex at 12000 rpm 2min. After the supernatant was discarded and the precipitation was fully dried, the EP tubes containing the precipitations were weighed(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 75% ethanol to dissolve salt and facilitate precipitation of protein. Then, we collected the precipitation at 12000rpm for 2min. After the supernatant was discarded and the precipitation was fully dried, the EP tubes containing the precipitations were weighed(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 know that the precipitation was a mixture of bacteria and mineralized products. So we can draw a conclusion that the bacteria and calcium carbonate precipitates could be cemented together

Result: The results showed a high variation rate, but we could still conclude that the precipitation was a mixture of bacteria and mineralized products.