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             <caption>Figure 1: LHyal-WT gene map</caption>
 
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             <caption>Figure 4: Construction of pPICZαA-LHyal-WT recombinant plasmid; A: Colony PCR to identify transformants; B: Plasmid map; C: Colony growth state; D: Gene sequencing map.</caption>
 
             <caption>Figure 4: Construction of pPICZαA-LHyal-WT recombinant plasmid; A: Colony PCR to identify transformants; B: Plasmid map; C: Colony growth state; D: Gene sequencing map.</caption>
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             <caption>Figure 5: Construction of pPICZαA-Lhyal-WT(op) recombinant plasmid; A: Colony PCR; B: Plasmid map; C: Colony growth; D: Gene sequencing.</caption>
 
             <caption>Figure 5: Construction of pPICZαA-Lhyal-WT(op) recombinant plasmid; A: Colony PCR; B: Plasmid map; C: Colony growth; D: Gene sequencing.</caption>
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         <img src="https://static.igem.wiki/teams/5070/bba-k5070001/10.jpg" width="50%" alt="Figure 10: Graph of fluorescence quantitative PCR results">
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             <caption>Figure 10: A: Amplification curve; B: T-test using GraphPad Prism 8; C: Gene expression analysis under different treatments.</caption>
 
             <caption>Figure 10: A: Amplification curve; B: T-test using GraphPad Prism 8; C: Gene expression analysis under different treatments.</caption>

Latest revision as of 05:38, 29 September 2024


pPICZαA-LHyal-WT

pPICZαA-LHyal-WT


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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 178


pPICZαA-LHyal-WT(op) Gene Documentation

Introduction

Based on BBa_K2593004 (LHyal-WT), we constructed a new plasmid, pPICZαA-LHyal-WT (BBa_K5070001), 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 and Engineering Principle

We chose the wild HAase gene LHyal-WT first to produce HAase, and we decided to use the plasmid pPICZαA to be the plasmid that carries the gene. This plasmid is common to the host Pichia pastoris (we use GS115). It is also easy to operate and provides a stable microbial environment for us high school students. For each step of preparation, we intend to use PCR or gene sequencing to test whether our results are reliable. We use SDS-PAGE, transparent circle test, and DNS to test the enzyme activity and productivity.

Figure 1: LHyal-WT gene map
Figure 1: LHyal-WT gene map

First, we extract and digest the pPICZαA plasmid using the enzymes EcoR1 and Sal1. Then, PCR is used to amplify the LHyal-WT gene sequence. We use homologous recombination to link the vector and gene sequence. Finally, the recombinant plasmid is successfully built, and heat shock is applied to E. coli DH5α.

Figure 2: pPICZαA-LHyal-WT plasmid map
Figure 2: pPICZαA-LHyal-WT plasmid map

Experimental Approach

Construction of recombinant plasmid

We synthesized the LHyal sequence plasmid containing the hyaluronidase expression gene as the template, and the LHyalWT-F1/LHyalWT-R1 primer was used to amplify the LHyal sequence. Using the codon-optimized hyaluronidase expression gene LHyal(op) sequence plasmid synthesized by our company as the template, the LHyal-WT-F1 /LHyal-WT(op)-R1 primer amplified the LHyal(op) sequence. As shown in Fig 3, LHyal and LHyal(op) sequences with a size of 1530bp were successfully amplified by PCR. Gel recovery was carried out to prepare for subsequent construction of recombinant plasmid. EcoRI and SalI were used to perform double enzyme digestion on the extracted plasmid skeleton of pPICZαA. The size of the linearized plasmid after enzyme digestion was about 3500bp.

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

Construction of homologous recombinant plasmid

We assembled the LHyal-WT and LHyal-WT(op) gene sequences with the linearized plasmid pPICZαA by homologous recombination. The recombinant plasmid was transformed into Escherichia coli DH5α by heat shock transformation and cultured at 37°C for 12-16 hours. Finally, we successfully cultured transformants on an LB-zeocin resistance medium, as shown in Fig 4.

Figure 4: Construction of pPICZαA-LHyal-WT recombinant plasmid
Figure 4: Construction of pPICZαA-LHyal-WT recombinant plasmid; A: Colony PCR to identify transformants; B: Plasmid map; C: Colony growth state; D: Gene sequencing map.

Primers were used to verify the successful construction of the recombinant plasmid by colony PCR on 5' AOX1/3' AOX1. As seen from Fig 4A and Fig 5A, 1945 bp bands were amplified by pPICZαA-LHyal-WT and pPICZαA-Lhyal-WT(op). These results indicated successful construction.

Figure 5: Construction of pPICZαA-Lhyal-WT(op) recombinant plasmid
Figure 5: Construction of pPICZαA-Lhyal-WT(op) recombinant plasmid; A: Colony PCR; B: Plasmid map; C: Colony growth; D: Gene sequencing.

Characterization/Measurement

Transformation of recombinant plasmids into yeast cells GS115

We linearized the constructed plasmids pPICZɑA-LHyal-WT and pPICZɑA-LHyal-WT(op) using SacI for transformation into yeast cells GS115. The digestion results are shown in Fig 6, and the clearest bands were selected for gel recycling to prepare for subsequent yeast transformation.

Figure 6: Linearized digests of pPICZɑA-LHyal-WT and pPICZɑA-LHyal-WT(op)
Figure 6: Linearized digests of pPICZɑA-LHyal-WT and pPICZɑA-LHyal-WT(op)

Subsequently, we transformed the linearized pPICZɑA-LHyal-WT and pPICZɑA-LHyal-WT(op) plasmids into yeast cells GS115 by chemical transformation for better growth in yeast. Several transformants were randomly picked from the resistance plate YPD-zeocin, pretreated with lysis buffer, and then verified with colony PCR using primers against 5'AOX1/3'AOX1. The amplified target band was 1945 bp, as shown in Figures 7A and 8A.

Figure 7: Transformation of pPICZɑA-LHyal-WT into yeast cells GS115
Figure 7: Transformation of pPICZɑA-LHyal-WT into yeast cells GS115; A: Colony PCR validation; B: YPD-zeocin plate growth.
Figure 8: Transformation of pPICZɑA-LHyal-WT(op) into yeast cells GS115
Figure 8: Transformation of pPICZɑA-LHyal-WT(op) into yeast cells GS115; A: Colony PCR validation; B: YPD-zeocin plate growth.

SDS-PAGE for Protein Expression

As shown in Figures 7 and 8, the obtained bands were 1945bp in size, indicating that the two recombinant plasmids were successfully transformed into yeast cells. The successfully transformed yeast cells were subjected to YPD shake flask fermentation. After 24 hours of incubation, the yeast cell precipitate was collected, ultrasonically broken, and subjected to SDS-PAGE protein gel validation, which was used to detect the protein expression, as shown in Fig 9. The size of the obtained protein was calculated to be 50kDa, indicating that the protein was successfully expressed.

Figure 9: SDS-PAGE detection of protein expression
Figure 9: SDS-PAGE detection of protein expression; A: pPICZɑA-LHyal-WT; B: pPICZɑA-LHyal-WT(op)

Real-time Fluorescence Quantitative PCR to Detect Gene Copy Number

The fluorescence quantitative PCR reaction system is shown in Table 1, with 3 parallels per sample. The internal reference gene was ARG4, and the primers for the internal reference gene were ARG4-f1/ARG4-r2. The target gene, LHyal-WT, and the primers were LHyalWT-qPCR-f1/LHyalWT-qPCR-r1, and the target gene LHyal-WT(op) and the primers were WT(OP)-qPCR-f3/WT(OP)-qPCR-r4.

Table 1: Fluorescence Quantitative PCR Reaction System

Ingredient Volume (μL)
2×SYBR green 10
Forward primer 0.5
Reverse primer 0.5
Genome templates 1
H2O 8

In Fig 10A, the amplification curve from real-time fluorescence quantitative PCR (qPCR) is shown. The experimental data were subjected to a t-test to compare the changes in the expression of the LHyal gene between the wild-type strain and the modified strain. As shown in Fig 10B, the results indicated a significant difference in gene expression between LHyal-WT and LHyal-WT(op) (P=0.0016<0.05).

Figure 10: Graph of fluorescence quantitative PCR results
Figure 10: A: Amplification curve; B: T-test using GraphPad Prism 8; C: Gene expression analysis under different treatments.

Determination of Recombinant Yeast Hyaluronidase Activity by DNS Method

The DNS (dinitro salicylic acid) method is a colorimetric method for determining the reducing sugar content. Hyaluronidase can produce glucose by decomposing hyaluronan, and we characterized hyaluronidase activity by the amount of glucose produced per unit of time. Before testing the samples' glucose content, a glucose standard curve was prepared, as shown in Table 2.

Table 2: Glucose Standard Curve Preparation

Serial number Glucose (mL) ddH2O (mL) DNS (mL) Total Volumetric (mL) Glucose (mg)
0 0 1 2 3 0
1 0.05 0.95 2 3 0.1
2 0.075 0.925 2 3 0.15
3 0.1 0.9 2 3 0.2
4 0.125 0.875 2 3 0.25
5 0.15 0.85 2 3 0.3
6 0.175 0.825 2 3 0.35
7 0.2 0.8 2 3 0.4

After preparing the glucose standard curve, we measured glucose content using the DNS method. The results are shown in Fig 11. The glucose content generated per unit of time helped us calculate enzyme activity. As seen in Fig 12, the optimized strain pPICZɑA-LHyal-WT(op) produced higher enzyme activity and glucose content than the wild-type strain pPICZɑA-LHyal-WT.

Figure 11: Glucose standard curve preparation
Figure 11: Glucose standard curve preparation
Figure 12: Determination of recombinant yeast hyaluronidase activity by DNS method
Figure 12: Determination of recombinant yeast hyaluronidase activity by DNS method; A: Enzyme activity; B: Glucose content over time.

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.