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<partinfo>BBa_K5070002 short</partinfo>

pPICZ&#945;A-LHyal-WT(op)



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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K5070002 SequenceAndFeatures</partinfo>

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    <title>High-Yield Hyaluronidase Pichia pastoris Cell Factory Construction</title>
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    <!-- Introduction Section -->
    <h2>Introduction</h2>
    <p>Based on BBa_K2593004 (LHyal-WT), we constructed a new plasmid, pPICZαA-LHyal-WT(OP) (BBa_K5070002), utilizing the pPICZαA vector and incorporating codon optimization techniques. The primary aim of codon optimization is to enhance the expression efficiency of HAase.</p>

    <!-- Construction Design Section -->
    <h2>Construction Design</h2>
    <p>The original leech hyaluronidase (HAase) possesses a naturally derived coding sequence. We applied codon optimization to mitigate the challenges of heterologous expression in <i>Pichia pastoris</i>. This strategy enhances the expression efficiency of functional leech HAase, thereby improving the yield rate. Furthermore, we used the pPICZαA as an expression vector, which introduced an α-factor secretion signal at the N-terminus to facilitate the secretion of mature leech HAase.</p>

    <!-- Figure 1 -->
    <div style="text-align:center;">
        <img src="https://static.igem.wiki/teams/5070/bba-k5070002/1.png" width="70%" alt="Figure 1: LHyal-WT(op) gene map">
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            <caption>Figure 1: LHyal-WT(op) gene map</caption>
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    <p>We chose the HAase gene LHyal-WT(op) to produce HAase and used the pPICZαA plasmid as the carrier. This plasmid is common for use in <i>Pichia pastoris</i> (GS115), making it accessible and stable for high school students. We employed PCR and gene sequencing for every preparation step to verify results. Additionally, we used SDS-PAGE and a transparent circle test to assess enzyme activity and productivity, and real-time fluorescence quantitative PCR and DNS to compare the productivity of WT and OP versions of the gene.</p>

    <!-- Engineering Principle Section -->
    <h3>Engineering Principle</h3>
    <p>First, we extracted and digested the pPICZαA plasmid using EcoR1 and Sal1. PCR was then used to amplify the LHyal-WT(op) gene sequence, followed by homologous recombination to link the vector and gene sequence. After successful plasmid construction, heat shock was used to transform <i>E. coli</i> DH5α.</p>

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        <img src="https://static.igem.wiki/teams/5070/bba-k5070002/2.png" width="50%" alt="Figure 2: pPICZαA-LHyal-WT(op) plasmid map">
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            <caption>Figure 2: pPICZαA-LHyal-WT(op) plasmid map</caption>
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    <!-- Cultivation, Protein Expression, and SDS-PAGE Section -->
    <h3>Cultivation, Protein Expression, and SDS-PAGE</h3>
    <p>We used gel electrophoresis to confirm gene amplification. The LHyal-WT(op) gene, which is 1470 bp, was clearly visible on the gel, indicating successful amplification.</p>

    <!-- Figure 3 -->
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        <img src="https://static.igem.wiki/teams/5070/bba-k5070002/3.png" width="50%" alt="Figure 3: PCR amplification and acquisition of target gene">
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            <caption>Figure 3: PCR amplification and acquisition of target gene; Amplification of LHyal-WT(op); Double digestion of pPICZαA plasmid.</caption>
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    <p>Once the recombinant plasmid was integrated into DH5α, we cultured it for one day, using antibiotics, PCR, and gene sequencing to confirm the correctness of the procedure. The results are shown in Figure 4.</p>

    <!-- Figure 4 -->
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        <img src="https://static.igem.wiki/teams/5070/bba-k5070002/4.png" width="70%" alt="Figure 4: Construction of pPICZαA-Lhyal-WT(op) recombinant plasmid">
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            <caption>Figure 4: A: Colony PCR; B: Plasmid map of pPICZαA-Lhyal-WT(op); C: Colony growth; D: Gene sequencing.</caption>
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    <p>We used enzymes to digest the recombinant plasmid for transformation into yeast. The linearized digests are shown in Figure 5.</p>

    <!-- Figure 5 -->
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        <img src="https://static.igem.wiki/teams/5070/bba-k5070002/5.png" width="40%" alt="Figure 5: Linearised digests of pPICZɑA-LHyal-WT(op)">
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            <caption>Figure 5: Linearised digests of pPICZɑA-LHyal-WT(op)</caption>
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    <p>We then transformed the recombinant plasmid into yeast and verified its sequence via PCR and gel electrophoresis. We also used antibiotics to ensure selection. The results are shown in Figure 6.</p>

    <!-- Figure 6 -->
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        <img src="https://static.igem.wiki/teams/5070/bba-k5070002/6.png" width="50%" alt="Figure 6: Transformation of pPICZɑA-LHyal-WT into yeast cells GS115">
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            <caption>Figure 6: A: Colony PCR validation; B: YPD-zeocin plate growth plot.</caption>
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    <p>We then used SDS-PAGE to test protein expression, with the HAase protein expressed at around 47 kDa. The enzyme activity was confirmed using a transparent circle test. The SDS-PAGE results are shown in Figure 7.</p>

    <!-- Figure 7 -->
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        <img src="https://static.igem.wiki/teams/5070/bba-k5070002/7.png" width="30%" alt="Figure 7: SDS-PAGE detection of protein expression">
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            <caption>Figure 7: SDS-PAGE detection of protein expression: pPICZɑA-LHyal-WT(op)</caption>
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    <!-- Characterization/Measurement Section -->
    <h3>Characterization/Measurement</h3>
    <p>HAase activity was measured after induction in both wild-type and optimized yeast strains. The optimized strain exhibited significantly higher HAase activity, suggesting that the optimization strategies enhanced enzyme production. Further analysis quantified these improvements using real-time fluorescence quantitative PCR to compare WT and OP productivity.</p>

    <!-- Figure 8 -->
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        <img src="https://static.igem.wiki/teams/5070/bba-k5070002/8.png" width="70%" alt="Figure 8: Graph of fluorescence quantitative PCR results">
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            <caption>Figure 8: A: Amplification curve; B: T-test using GraphPad Prism 8; C: Gene expression analysis under different treatments, P=0.0016<0.05.</caption>
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    <p>Enzyme activity was further measured through a transparent circle test. HAase activity produces glucose upon degrading HA, and the DNS method quantified the glucose produced. A glucose standard curve was sketched to show the expected glucose amount. Yeast was cultivated for 0h, 24h, 48h, 72h, and 96h, and productivity was shown in bar charts, as in Figure 9.</p>

    <!-- Figure 9 -->
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        <img src="https://static.igem.wiki/teams/5070/bba-k5070002/9.jpg" width="50%" alt="Figure 9: Glucose standard curve preparation">
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            <caption>Figure 9: Glucose standard curve preparation</caption>
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    <p>To further conclude the experiment, we used DNS over a 96-hour period to track HAase activity. Initially, the activity of HAase produced by WT was higher than that of OP, but after prolonged induction, the activity of OP exceeded that of WT, as shown in Figure 10.</p>

    <!-- Figure 10 -->
    <div style="text-align:center;">
        <img src="https://static.igem.wiki/teams/5070/bba-k5070002/10.jpg" width="50%" alt="Figure 10: Determination of recombinant yeast hyaluronidase activity by DNS method">
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            <caption>Figure 10: Determination of recombinant yeast hyaluronidase activity by DNS method</caption>
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    <!-- Summary Section -->
        <h3>Summary</h3>
    <p>The iGEM project "High-Yield Hyaluronidase Pichia pastoris Cell Factory Construction" addresses the challenges associated with the costly and limited production of hyaluronidase (HAase), an enzyme extensively used in medical applications. This project involves genetically modifying <i>Pichia pastoris</i> to produce HAase derived from leeches. Key activities include the design and synthesis of the HAase gene, the creation of expression plasmids, and the transformation of <i>Pichia pastoris</i> with these plasmids. Strains with high-copy integrations are selected through antibiotic resistance.</p>

    <p>Enzyme activity is analyzed using SDS-PAGE and DNS methods to optimize production and correlate gene copy number with HAase activity. The ultimate goal is to develop a genetically engineered yeast strain that can produce HAase efficiently, potentially impacting the biopharmaceutical industry by offering a more cost-effective and scalable production method for hyaluronidase.</p>

    <!-- References Section -->
    <h3>References</h3>
    <p>[1] Cui, Y., et al. (2015). High-level expression of human hyaluronidase in <i>Pichia pastoris</i> and its potential application in tumour therapy. Applied Microbiology and Biotechnology, 99(21), 8817-8827.</p>
    <p>[2] Liu, C., et al. (2017). Enhanced production of recombinant human hyaluronidase in <i>Pichia pastoris</i> by optimizing codon usage and fermentation conditions. Biotechnology Letters, 39(8), 1233-1239.</p>
    <p>[3] Wang, J., et al. (2018). A novel method for quantitative detection of hyaluronidase activity using a DNS assay. Biotechnology Progress, 34(2), 434-440.</p>
    <p>[4] Zhang, J., et al. (2019). Metabolic engineering of <i>Pichia pastoris</i> for the production of biopharmaceuticals. Microbial Cell Factories, 18(1), 1-12.</p>
    <p>[5] Chen, X., et al. (2020). Synthetic biology approaches to enhance the production of heterologous proteins in yeast. Biotechnology Journal, 15(5), 1-15.</p>

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