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    <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>
  
 
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Latest revision as of 11:03, 30 September 2024


pPICZαA-LHyal-WT(op)

pPICZαA-LHyal-WT(op)


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 715
  • 1000
    COMPATIBLE WITH RFC[1000]

High-Yield Hyaluronidase Pichia pastoris Cell Factory Construction

Introduction

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.

Construction Design

The original leech hyaluronidase (HAase) possesses a naturally derived coding sequence. We applied codon optimization to mitigate the challenges of heterologous expression in Pichia pastoris. 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.

Figure 1: LHyal-WT(op) gene map
Figure 1: LHyal-WT(op) gene map

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 Pichia pastoris (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.

Engineering Principle

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 E. coli DH5α.

Figure 2: pPICZαA-LHyal-WT(op) plasmid map
Figure 2: pPICZαA-LHyal-WT(op) plasmid map

Cultivation, Protein Expression, and SDS-PAGE

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.

Figure 3: PCR amplification and acquisition of target gene
Figure 3: PCR amplification and acquisition of target gene; Amplification of LHyal-WT(op); Double digestion of pPICZαA plasmid.

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.

Figure 4: Construction of pPICZαA-Lhyal-WT(op) recombinant plasmid
Figure 4: A: Colony PCR; B: Plasmid map of pPICZαA-Lhyal-WT(op); C: Colony growth; D: Gene sequencing.

We used enzymes to digest the recombinant plasmid for transformation into yeast. The linearized digests are shown in Figure 5.

Figure 5: Linearised digests of pPICZɑA-LHyal-WT(op)
Figure 5: Linearised digests of pPICZɑA-LHyal-WT(op)

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.

Figure 6: Transformation of pPICZɑA-LHyal-WT into yeast cells GS115
Figure 6: A: Colony PCR validation; B: YPD-zeocin plate growth plot.

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.

Figure 7: SDS-PAGE detection of protein expression
Figure 7: SDS-PAGE detection of protein expression: pPICZɑA-LHyal-WT(op)

Characterization/Measurement

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.

Figure 8: Graph of fluorescence quantitative PCR results
Figure 8: A: Amplification curve; B: T-test using GraphPad Prism 8; C: Gene expression analysis under different treatments, P=0.0016<0.05.

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.

Figure 9: Glucose standard curve preparation
Figure 9: Glucose standard curve preparation

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.

Figure 10: Determination of recombinant yeast hyaluronidase activity by DNS method
Figure 10: Determination of recombinant yeast hyaluronidase activity by DNS method

Summary

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 Pichia pastoris 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 Pichia pastoris with these plasmids. Strains with high-copy integrations are selected through antibiotic resistance.

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.

References

[1] Cui, Y., et al. (2015). High-level expression of human hyaluronidase in Pichia pastoris and its potential application in tumour therapy. Applied Microbiology and Biotechnology, 99(21), 8817-8827.

[2] Liu, C., et al. (2017). Enhanced production of recombinant human hyaluronidase in Pichia pastoris by optimizing codon usage and fermentation conditions. Biotechnology Letters, 39(8), 1233-1239.

[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.

[4] Zhang, J., et al. (2019). Metabolic engineering of Pichia pastoris for the production of biopharmaceuticals. Microbial Cell Factories, 18(1), 1-12.

[5] Chen, X., et al. (2020). Synthetic biology approaches to enhance the production of heterologous proteins in yeast. Biotechnology Journal, 15(5), 1-15.