Difference between revisions of "Part:BBa K4202006"

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<p>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.</p>
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<p><b>Result:</b> 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.</p>
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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 <i>Bacillus subtilis</i> with CA(<partinfo>BBa_K4202004</partinfo>), EutM-SpyCatcher(<partinfo>BBa_K4202015</partinfo>), Hag-SpyTag588 were cultured in 50 ml LB medium at 25 <sup>o</sup>C, 220 rpm, pH 8.0 for 4 days. At the first day and the third day 500μl 1M CaCl<sub>2</sub> was added into the medium. Besides, a group without the <i>Bacillus subtilis</i> containing CA was settled for control.</p>
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<p>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.</p>
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<div align="center">[[File:YZH-167.png|600px]]</div>
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<div align="center"><b>Fig 2</b> 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.</div>
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<div align="center">[[File:YZH-168.png|600px]]</div>
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<div align="center"><b>Fig 3</b> The image of the confocal microscope(FLUOVIEW FV3000). A: SYTO; B: Photo without Ex; C: Merge.</div>
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<p>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. </p>
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<p>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.</p>
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<p>Step 2: We collected the complex at 12000 rpm 2min. The surnatant was discarded, fully dried and weighed to obtain M2.</p>
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<p>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.</p>
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<p>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.</p>
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<p>Step 5: Calculate the yield according to the following formula.
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<br>A1=(M2-M1 ) / V * 100%
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<br>A2=(M3-M2) / V * 100%</p>
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<div align="center">[[File:YZH-169.png|600px]]</div>
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<p><b>Result:</b> The results showed a high variation rate, but we could still conclude that the precipitation was a mixture of bacteria and mineralized products.</p>

Revision as of 04:14, 12 October 2022


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.


YZH-166.png
Fig 1 The result of fluorescence confocal microscope


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. Besides, a group without the Bacillus subtilis containing CA was settled for control.</p>

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.

YZH-167.png
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.
YZH-168.png
Fig 3 The image of the confocal microscope(FLUOVIEW FV3000). A: SYTO; B: Photo without Ex; C: Merge.


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%

YZH-169.png

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