Difference between revisions of "Part:BBa K5531000"
 
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Rh3C-P15VP4
 
  
  
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===Usage and Biology===
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===Functional Parameters===
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    <h2>BBa_K5531000 (Rh3C-P15VP4)</h2>
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    <h3>Profile</h3>
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        <strong>Name:</strong> Rh3C-P15VP4<br>
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        <strong>Base Pairs:</strong> 2329 bp<br>
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        <strong>Origin:</strong> Human Type III Collagen; synthesized<br>
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        <strong>Properties:</strong> Rh3C-P15VP4 contains humanized type III collagen fused with the VP4 protein from the rotavirus with serotype P15.
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    </p>
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    <h3>Usage and Biology</h3>
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    <p>
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        Rh3C-P15VP4 contains humanized type III collagen fused with the VP4 protein from the rotavirus with serotype P15. Recombinant humanized type III collagen (Rh3C) was found to have stronger cell adhesion properties, suggesting a more stable triple helix conformation. Since type III collagen is widely distributed in the human body, it is more difficult to generate an immune response in the human body and can effectively form trimers [1].
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        <img src="https://static.igem.wiki/teams/5531/bba-k5531000/1.png" alt="Figure 1: Rh3C-P15VP4 Gene map">
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        <div class="caption">Fig. 1. Rh3C-P15VP4 Gene map</div>
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    <h3>Experimental Approach</h3>
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        We isolated pET-Dual-N-His-TEV vectors from bacterial solutions primarily by centrifugation. The vectors were then obtained from the remains in the absorption column. Subsequently, we linearized the vectors using restriction enzymes and conducted electrophoresis to analyze the products. The electrophoresis result displayed correctness toward the expected outcome (HisRh3C-P15VP4 is 2000 bp, and P4H is 755 bp). We selected colonies and sent them directly for sequencing. Fig. 2C shows the success in pET-Dual-HisRh3C-P15VP4-P4H construction.
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        <img src="https://static.igem.wiki/teams/5531/bba-k5531000/2.jpg" alt="Figure 2: The results of pET-Dual-HisRh3C-P15VP4-P4H">
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        <div class="caption">Fig. 2. The results of pET-Dual-HisRh3C-P15VP4-P4H</div>
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        Later, the plasmid mounted with the gene of Rh3C-P15VP4 was transferred to <em>E. coli</em> DH5α to replicate. The extracted plasmid was transferred into <em>E. coli</em> BL21, which can help express His-Rh3C-P15VP4. After the colony PCR of <em>E. coli</em> BL21 was finished and verified, the bacteria were cultured and treated with 0.2 mM IPTG, which can promote protein expression. After promoting the protein expression overnight, the protein was purified via His-tag Purification Resin and went through SDS-PAGE electrophoresis and Native-PAGE electrophoresis. The target protein Rh3C-P15VP4 has a size of 85 kDa (Figure 3).
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        <img src="https://static.igem.wiki/teams/5531/bba-k5531000/3.jpg" alt="Figure 3: The expression of His-Rh3C-P15VP4 using E. coli BL21. SDS-PAGE gels showing the purification results.">
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        <div class="caption">Fig. 3. The expression of His-Rh3C-P15VP4 using E. coli BL21. SDS-PAGE gels showing the purification results. The loading sequence is protein marker, whole cell lysate, precipitate, supernatant, flow-through, unwanted proteins, and target protein.</div>
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    <h3>References</h3>
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    <p>
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        [1] Jalan AA, Demeler B, Hartgerink JD. Hydroxyproline-free single-composition ABC collagen heterotrimer. <em>J Am Chem Soc</em>. 2013 Apr 24;135(16):6014-7. doi: 10.1021/ja402187t.
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Latest revision as of 03:51, 30 September 2024


Rh3C-P15VP4



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1181
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


BBa_K5531000 (Rh3C-P15VP4)

BBa_K5531000 (Rh3C-P15VP4)

Profile

Name: Rh3C-P15VP4
Base Pairs: 2329 bp
Origin: Human Type III Collagen; synthesized
Properties: Rh3C-P15VP4 contains humanized type III collagen fused with the VP4 protein from the rotavirus with serotype P15.

Usage and Biology

Rh3C-P15VP4 contains humanized type III collagen fused with the VP4 protein from the rotavirus with serotype P15. Recombinant humanized type III collagen (Rh3C) was found to have stronger cell adhesion properties, suggesting a more stable triple helix conformation. Since type III collagen is widely distributed in the human body, it is more difficult to generate an immune response in the human body and can effectively form trimers [1].

Figure 1: Rh3C-P15VP4 Gene map
Fig. 1. Rh3C-P15VP4 Gene map

Experimental Approach

We isolated pET-Dual-N-His-TEV vectors from bacterial solutions primarily by centrifugation. The vectors were then obtained from the remains in the absorption column. Subsequently, we linearized the vectors using restriction enzymes and conducted electrophoresis to analyze the products. The electrophoresis result displayed correctness toward the expected outcome (HisRh3C-P15VP4 is 2000 bp, and P4H is 755 bp). We selected colonies and sent them directly for sequencing. Fig. 2C shows the success in pET-Dual-HisRh3C-P15VP4-P4H construction.

Figure 2: The results of pET-Dual-HisRh3C-P15VP4-P4H
Fig. 2. The results of pET-Dual-HisRh3C-P15VP4-P4H

Later, the plasmid mounted with the gene of Rh3C-P15VP4 was transferred to E. coli DH5α to replicate. The extracted plasmid was transferred into E. coli BL21, which can help express His-Rh3C-P15VP4. After the colony PCR of E. coli BL21 was finished and verified, the bacteria were cultured and treated with 0.2 mM IPTG, which can promote protein expression. After promoting the protein expression overnight, the protein was purified via His-tag Purification Resin and went through SDS-PAGE electrophoresis and Native-PAGE electrophoresis. The target protein Rh3C-P15VP4 has a size of 85 kDa (Figure 3).

Figure 3: The expression of His-Rh3C-P15VP4 using E. coli BL21. SDS-PAGE gels showing the purification results.
Fig. 3. The expression of His-Rh3C-P15VP4 using E. coli BL21. SDS-PAGE gels showing the purification results. The loading sequence is protein marker, whole cell lysate, precipitate, supernatant, flow-through, unwanted proteins, and target protein.

References

[1] Jalan AA, Demeler B, Hartgerink JD. Hydroxyproline-free single-composition ABC collagen heterotrimer. J Am Chem Soc. 2013 Apr 24;135(16):6014-7. doi: 10.1021/ja402187t.