Difference between revisions of "Part:BBa K5384007"
 
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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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     <div class="image-name">Figure 1: </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 tags (BBa_K5384006), the Vglycin gene (BBa_K5384001), the Enterokinase recognition site (BBa_K5384002), the Asp-pro acid-sensitive site (BBa_K5384003), and upstream the AOX1 promoter (BBa_K5384004), and α-secretion signal peptide (BBa_K5384005).
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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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===Application===
 
===Application===
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After inserting the VGLYCIN-encoding polypeptide gene from the Picot yeast plasmid, the VGLYCIN gene and the DDDDK site were ligated to the plasmid pPIC9K. We designed new plasmids in our experiments because we needed to transform Pichia yeast to express our target protein VGLYCIN, and we constructed and used the plasmid PIC9K-3His-DDDK-vg with the replication start of the Pichia yeast plasmid, AOX1 promoter promoter, α-factor secretion signal replication signal, and plasmid AOX1 terminator replication terminator, which facilitates insertion and expression of target genes and screening of recombinants. The Ori site of the Pasteur Picot yeast plasmid was derived from the Pasteur Picot yeast plasmid.
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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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===Functional Parameters===
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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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[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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[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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[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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[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.  
 
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).
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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).