Part:BBa_K4202006

This part is a fusion protein of Hag and SpyTag , and it`s used for the bio-scafford.

In this protein, SpyTag is inserted at a specific site for presentation of the epitope on the flagellar surface after assembly. In addition, we replaced amino acid at 222 with Cys ,which can be reacted with maleimide. In our experiments , we will use the SpyCatcher-mRFP , maleimide and other fluorescent dye to characterize this protein by fluorescence fluorescence microscope.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 568
  

Result

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 and PHY-EutM-SpyCatcher(BBa_K4202015) 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 1 Confocal laser scanning microscope of different groups. The cells were stained by SYTO for indicated.

Result: 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-SpyCatcher(BBa_K4202015), Hag-SpyTag588 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 2 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 3 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.