Difference between revisions of "Part:BBa K5375003"

 
 
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<partinfo>BBa_K5375003 short</partinfo>
 
<partinfo>BBa_K5375003 short</partinfo>
  
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pPICZ&#945;A-HSP70-sfGFP
 
  
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K5375003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5375003 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display  
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K5375003 parameters</partinfo>
 
<partinfo>BBa_K5375003 parameters</partinfo>
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__TOC__
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<span id="origin"></span>
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= Origin =
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 +
Synthesized by company and constructed by the team.
 +
 
 +
<span id="properties"></span>
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= Properties =
 +
 
 +
Fusion expression of protein HSP70-GFP.
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 +
<span id="usage-and-biology"></span>
 +
= Usage and Biology =
 +
 
 +
The pPICZαA vectors are 3.6 kb plasmids designed for the expression and secretion of recombinant proteins in *Pichia pastoris* yeast. These recombinant proteins are expressed as N-terminal fusions with the coding sequence of the α-factor secretion signal derived from *Saccharomyces cerevisiae*. The vectors facilitate high-level gene expression induced by methanol in *Pichia pastoris* and can be utilized with any strain of this yeast. We inserted the HSP70 coding gene into this plasmid for fusion expression with GFP.
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<span id="cultivation-purification-sds-page"></span>
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= Cultivation, Purification and SDS-PAGE =
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<html>
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<div style="text-align:center;">
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    <img src="https://static.igem.wiki/teams/5375/bba-k5375003/1.png" width="50%" style="display:block; margin:auto;" alt="plasmid diagram of pPICZαA-HSP70-sfGFP" >
 +
    <div style="text-align:center;">
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        <caption>Figure 1. Plasmid diagram of pPICZαA-HSP70-sfGFP.</caption>
 +
    </div>
 +
</div>
 +
</html>
 +
 
 +
The vector pPICZαA originates from the *Pichia pastoris* expression vector. It is methanol-inducible and allows high-level expression and secretion of recombinant proteins, specifically in yeast. This vector was chosen to measure the DNA expression of HSP70 in yeast.
 +
 
 +
<html>
 +
<div style="text-align:center;">
 +
    <img src="https://static.igem.wiki/teams/5375/bba-k5375003/2.png" width="50%" style="display:block; margin:auto;" alt="PCR amplification of the fragment used for plasmid construction" >
 +
    <div style="text-align:center;">
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        <caption>Figure 2. PCR amplification of the fragment used for plasmid construction.</caption>
 +
    </div>
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</div>
 +
</html>
 +
 
 +
We constructed pPICZαA-HSP70-sfGFP using homologous recombination. The HSP70-sfGFP sequence was amplified by PCR with a length of 2123 bp.
 +
 
 +
<html>
 +
<div style="text-align:center;">
 +
    <img src="https://static.igem.wiki/teams/5375/bba-k5375003/3.png" width="50%" style="display:block; margin:auto;" alt="Growth of plasmid pPICZaαA-HSP70 transformed bacteria on LB agar plates" >
 +
    <div style="text-align:center;">
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        <caption>Figure 3. Growth of plasmid pPICZaαA-HSP70 transformed bacteria on LB agar plates.</caption>
 +
    </div>
 +
</div>
 +
</html>
 +
 
 +
Secondly, the correct plasmids of pPICZαA-HSP70-sfGFP were transferred into yeast. They were cultured on YPD medium agar plates overnight. The image shows significant bacterial growth on YPD agar plates with easily observable individual colonies.
 +
 
 +
<html>
 +
<div style="text-align:center;">
 +
    <img src="https://static.igem.wiki/teams/5375/bba-k5375003/4.png" width="50%" style="display:block; margin:auto;" alt="Growth of pPICZαA-HSP70-sfGFP in yeast on YPD agar plates" >
 +
    <div style="text-align:center;">
 +
        <caption>Figure 4. Growth of pPICZαA-HSP70-sfGFP in yeast on YPD agar plates.</caption>
 +
    </div>
 +
</div>
 +
</html>
 +
 
 +
Finally, the protein expression of pPICZαA-HSP70 was tested by SDS-PAGE. The results are shown below, displaying the target band for HSP70 (78 kDa).
 +
 
 +
<html>
 +
<div style="text-align:center;">
 +
    <img src="https://static.igem.wiki/teams/5375/bba-k5375003/5.png" width="50%" style="display:block; margin:auto;" alt="SDS-PAGE analysis of HSP70 expression in yeast cells" >
 +
    <div style="text-align:center;">
 +
        <caption>Figure 5. SDS-PAGE analysis of HSP70 expression in yeast cells.</caption>
 +
    </div>
 +
</div>
 +
</html>
 +
 
 +
<span id="measurement-characterization"></span>
 +
= Measurement and Characterization =
 +
 
 +
We used DNA sequencing to determine the full nucleotide sequence of our reconstructed plasmids, verifying the accuracy and integrity of the pPICZαA-HSP70-sfGFP plasmids.
 +
 
 +
<html>
 +
<div style="text-align:center;">
 +
    <img src="https://static.igem.wiki/teams/5375/bba-k5375003/6.png" width="50%" style="display:block; margin:auto;" alt="Sanger sequencing result of pPICZαA-HSP70-sfGFP plasmid" >
 +
    <div style="text-align:center;">
 +
        <caption>Figure 6. Sanger sequencing result of pPICZαA-HSP70-sfGFP plasmid.</caption>
 +
    </div>
 +
</div>
 +
</html>
 +
 
 +
<span id="reference"></span>
 +
= Reference =
 +
 
 +
[https://www.thermofisher.cn/order/catalog/product/cn/zh/V19520](https://www.thermofisher.cn/order/catalog/product/cn/zh/V19520)
 +
 
 +
[https://www.snapgene.com/plasmids/yeast_plasmids/pPICZ(alpha)_](https://www.snapgene.com/plasmids/yeast_plasmids/pPICZ(alpha)_)

Latest revision as of 11:36, 25 September 2024

pPICZαA-HSP70-sfGFP



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 383
    Illegal BglII site found at 1307
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 468
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 358
    Illegal SapI.rc site found at 1677
    Illegal SapI.rc site found at 1966


Origin

Synthesized by company and constructed by the team.

Properties

Fusion expression of protein HSP70-GFP.

Usage and Biology

The pPICZαA vectors are 3.6 kb plasmids designed for the expression and secretion of recombinant proteins in *Pichia pastoris* yeast. These recombinant proteins are expressed as N-terminal fusions with the coding sequence of the α-factor secretion signal derived from *Saccharomyces cerevisiae*. The vectors facilitate high-level gene expression induced by methanol in *Pichia pastoris* and can be utilized with any strain of this yeast. We inserted the HSP70 coding gene into this plasmid for fusion expression with GFP.

Cultivation, Purification and SDS-PAGE

plasmid diagram of pPICZαA-HSP70-sfGFP
Figure 1. Plasmid diagram of pPICZαA-HSP70-sfGFP.

The vector pPICZαA originates from the *Pichia pastoris* expression vector. It is methanol-inducible and allows high-level expression and secretion of recombinant proteins, specifically in yeast. This vector was chosen to measure the DNA expression of HSP70 in yeast.

PCR amplification of the fragment used for plasmid construction
Figure 2. PCR amplification of the fragment used for plasmid construction.

We constructed pPICZαA-HSP70-sfGFP using homologous recombination. The HSP70-sfGFP sequence was amplified by PCR with a length of 2123 bp.

Growth of plasmid pPICZaαA-HSP70 transformed bacteria on LB agar plates
Figure 3. Growth of plasmid pPICZaαA-HSP70 transformed bacteria on LB agar plates.

Secondly, the correct plasmids of pPICZαA-HSP70-sfGFP were transferred into yeast. They were cultured on YPD medium agar plates overnight. The image shows significant bacterial growth on YPD agar plates with easily observable individual colonies.

Growth of pPICZαA-HSP70-sfGFP in yeast on YPD agar plates
Figure 4. Growth of pPICZαA-HSP70-sfGFP in yeast on YPD agar plates.

Finally, the protein expression of pPICZαA-HSP70 was tested by SDS-PAGE. The results are shown below, displaying the target band for HSP70 (78 kDa).

SDS-PAGE analysis of HSP70 expression in yeast cells
Figure 5. SDS-PAGE analysis of HSP70 expression in yeast cells.

Measurement and Characterization

We used DNA sequencing to determine the full nucleotide sequence of our reconstructed plasmids, verifying the accuracy and integrity of the pPICZαA-HSP70-sfGFP plasmids.

Sanger sequencing result of pPICZαA-HSP70-sfGFP plasmid
Figure 6. Sanger sequencing result of pPICZαA-HSP70-sfGFP plasmid.

Reference

[1](https://www.thermofisher.cn/order/catalog/product/cn/zh/V19520)

[2](https://www.snapgene.com/plasmids/yeast_plasmids/pPICZ(alpha)_)