Difference between revisions of "Part:BBa K5384007"
 
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<partinfo>BBa_K5384007 short</partinfo>
 
<partinfo>BBa_K5384007 short</partinfo>
  
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VGLYCIN gene and DDDDK site were linked to plasmid pPIC9K after inserting VGLYCIN coding polypeptide gene from Pichia pastoris plasmid. We designed the new plasmid in the experiment, because we need to transform Pichia pastoris to express our target protein VGLYCIN, the plasmid pPIC9K-3His-DDDK-vg, which we constructed and used, has a replication origin of the Pichia pastoris plasmid, an AOX1 promotor promoter, an &#945;-factor secretion signal replication signal, and a plasmid AOX1 terminator replication terminator, it is convenient to insert and express the target gene and screen out the recombinant. The Ori site of the Pichia pastoris plasmid was derived from the Pichia pastoris plasmid.
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Vglycin gene and DDDDK were inserted to the plasmid pPIC9K ang then transformed into Pichia pastoris. We designed the new plasmid in the experiment to massively produce Vg. The plasmid pPIC9K-His-DDDDK-Vg, which we constructed and used, has an AOX 1 promoter, an α-factor secretion signal, and a plasmid AOX 1 terminator, it is convenient to insert and express the target gene and purify the recombinant.  
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<html>
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  .image-box {
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        text-align: center;
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        margin: 0px auto;
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  .image-container img {
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        <img src="https://static.igem.wiki/teams/5384/part-ku/part-7-1.png">
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    </div>
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    <div class="image-name">Figure 1 Visualization of the pPIC9K-His-DDDDK-Vg for overexpression</div>
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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This composite part contains three His-tag (BBa_K5384006), the Vglycin gene (BBa_K5384001), the Enterokinase recognition site (BBa_K5384002), the Asp-pro acid-sensitive site (BBa_K5384003), and the AOX 1 promoter (BBa_K5384004), and α-secretion signal peptide (BBa_K5384005).
  
 
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<span class='h3bb'>Sequence and Features</span>
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===Sequence and Features===
 
<partinfo>BBa_K5384007 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5384007 SequenceAndFeatures</partinfo>
  
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<partinfo>BBa_K5384007 parameters</partinfo>
 
<partinfo>BBa_K5384007 parameters</partinfo>
 
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===Application===
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After inserting the Vglycin-encoding polypeptide gene into the plasmid, we transformed the plasmid into Pichia pastoris to produce Vg. The plasmid pPIC9K-His-DDDDK-Vg we constructed and used has AOX 1 promoter, α-factor secretion signal, and plasmid AOX 1 terminator, which facilitates insertion and expression of target genes and purification of recombinants.
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===References===
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[1] WANG Wenli, WANG Yunlong, LI Chenyang, et al. Preparation, Identification and Preliminary Application of His-tagged Monoclonal Antibody[J]. J Cell and Molecular Immunol,2008,24(4):399-400. DOI:10.3321/j.issn:1007-8738.2008.04.028.
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[2] LI Shuying, ZHAO Zhonglin, NIE Ying, et al. Research Progress on Nattokinase[J]. China Agricultural Science and Technology Review,2013,15(4):139-143.] DOI:10.3969/j.issn.1008-0864.2013.04.21.
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[3] Huang Zhili, Luo Lixin, Yang Rude, et al. Nattokinase[J]. Chemistry of Life,2000(2):82-83.] DOI:10.3969/j.issn.1000-1336.2000.02.012.
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[4] Zhao Fuyong, Yan Han, Ren Guangxu, et al. Research Progress of Recombinant Nattokinase[J]. China Food and Nutrition,2019,25(7):41-45.] DOI:10.3969/j.issn.1006-9577.2019.07.008.
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Establishment of nattokinase detection system and a preliminary study on its transmembrane transport pathway[J]. Journal of Chengdu Medical College,2016,11(5):532-536,564. DOI:10.3969/j.issn.1674-2257.2016.05.003.
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[5] Tang Xiaoyan. Screening and application evaluation of efficient transcriptional termination sequences in Pichia pastoris[D]. Guangdong:South China University of Technology,2019(in Chinese).

Latest revision as of 14:20, 1 October 2024


pPIC9K-His-DDDDK-Vg

Vglycin gene and DDDDK were inserted to the plasmid pPIC9K ang then transformed into Pichia pastoris. We designed the new plasmid in the experiment to massively produce Vg. The plasmid pPIC9K-His-DDDDK-Vg, which we constructed and used, has an AOX 1 promoter, an α-factor secretion signal, and a plasmid AOX 1 terminator, it is convenient to insert and express the target gene and purify the recombinant.

Figure 1 Visualization of the pPIC9K-His-DDDDK-Vg for overexpression

Usage and Biology

This composite part contains three His-tag (BBa_K5384006), the Vglycin gene (BBa_K5384001), the Enterokinase recognition site (BBa_K5384002), the Asp-pro acid-sensitive site (BBa_K5384003), and the AOX 1 promoter (BBa_K5384004), and α-secretion signal peptide (BBa_K5384005).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1121
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1121
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 142
    Illegal XhoI site found at 1342
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1121
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1121
  • 1000
    COMPATIBLE WITH RFC[1000]


Application

After inserting the Vglycin-encoding polypeptide gene into the plasmid, we transformed the plasmid into Pichia pastoris to produce Vg. The plasmid pPIC9K-His-DDDDK-Vg we constructed and used has AOX 1 promoter, α-factor secretion signal, and plasmid AOX 1 terminator, which facilitates insertion and expression of target genes and purification of recombinants.

References

[1] WANG Wenli, WANG Yunlong, LI Chenyang, et al. Preparation, Identification and Preliminary Application of His-tagged Monoclonal Antibody[J]. J Cell and Molecular Immunol,2008,24(4):399-400. DOI:10.3321/j.issn:1007-8738.2008.04.028.

[2] LI Shuying, ZHAO Zhonglin, NIE Ying, et al. Research Progress on Nattokinase[J]. China Agricultural Science and Technology Review,2013,15(4):139-143.] DOI:10.3969/j.issn.1008-0864.2013.04.21.

[3] Huang Zhili, Luo Lixin, Yang Rude, et al. Nattokinase[J]. Chemistry of Life,2000(2):82-83.] DOI:10.3969/j.issn.1000-1336.2000.02.012.

[4] Zhao Fuyong, Yan Han, Ren Guangxu, et al. Research Progress of Recombinant Nattokinase[J]. China Food and Nutrition,2019,25(7):41-45.] DOI:10.3969/j.issn.1006-9577.2019.07.008. Establishment of nattokinase detection system and a preliminary study on its transmembrane transport pathway[J]. Journal of Chengdu Medical College,2016,11(5):532-536,564. DOI:10.3969/j.issn.1674-2257.2016.05.003.

[5] Tang Xiaoyan. Screening and application evaluation of efficient transcriptional termination sequences in Pichia pastoris[D]. Guangdong:South China University of Technology,2019(in Chinese).