Plasmid

Part:BBa_K4870017

Designed by: Ding Xuebin   Group: iGEM23_Fujian-United   (2023-07-18)


pYES2-Hyg-GAL1p-An_phy33-SED1-RPL41Bt

Contribution: BBa_K4870017

Construction Design

We obtained a series of ancestral enzyme sequences of E. coli phytase AppA at the website (https://loschmidt.chemi.muni.cz/fireprotasr/). Prediction using DeepSTABp (https://csb-deepstabp.bio.rptu.de/) revealed that the An_phy33 sequence had the highest melting temperature Tm of 87 °C compared to 51 °C for wild-type AppA, so we chose to use this sequence for this round of experiments.

Similar to the previous round of expression frames, we linked the yeast promoter GAL, MF-alpha-1 signal peptide, An_phy33 gene, yeast surface anchoring protein gene SED1, and RPL41Bt yeast (Figure 1). Through restriction endonuclease digestion and linkage, we constructed the An_phy33 display plasmid: pYES2-Hyg-GAL1p-α-An_phy33-SED1-RPL41Bt.

Figure 1
Figure 1: Expression frame of the An_phy33 display plasmid

Engineering Principle

We transformed the plasmid into yeast and fused the phytase encoding gene with the anchoring protein encoding gene on the cell surface to produce yeast single cell protein with phytase activity, which can be added to feed to enhance animal nutrition and absorption[1],[2].

Figure 2
Figure 2: Design diagram of this project. Image by LIU JINLE.

Experimental Approach

To construct the plasmid pYES2-Hyg-GAL1p-α-An_phy33-SED1-RPL41Bt, we used a synthetic plasmid containing the An_phy33 sequence as a template to amplify An_phy33 sequence by PCR. Then, the An_phy33 sequence was inserted into the SphⅠ and AvrⅡ sites of plasmid pYES2-Hyg-GAL1p-α-SED1-RPL41Bt, by restriction endonuclease digestion and linkage.

Figure 3
Figure 3: Construction of the An_phy33 display plasmid

The plasmid was extracted from the positive transformant and linearized. It was then transformed into BY4741 yeast-competent cells and incubated at 30 °C for 48 h. Colony PCR and sequencing verified the correct integration and sequence of the An_phy33 fragment in yeast.

Figure 4
Figure 4: Results of An_phy33 display plasmid transformation of yeast cells. A positive yeast transformant was cultured to a logarithmic growth period. Galactose induction yielded cells displaying phytase on the surface. Then, the cells were pelleted, washed with 50 mM sodium acetate buffer (pH 5.0), and lysed. Lysate centrifugation gave a supernatant and precipitate containing cell wall-bound phytase. The SDS-PAGE result showed a successful surface display of the An_phy33 phytase (Figure 5).

Phytase activity assays were performed on surface-displayed and cell wall-bound phytase. Lysed phytase precipitate exhibited higher activity and was used to determine phytase activity under different pH and temperature conditions. As shown in Figure 9, the activity of the An_phy33 phytase is higher than that of the AppA phytase. It reaches maximum activity at a pH value of 6 or a reaction temperature of 55 °C.

Figure 5
Figure 5: Expression and enzyme activity test results of An_phy33.

References

  1. Chen X, Xiao Y, Shen W, Govender A, Zhang L, Fan Y, Wang Z. Display of phytase on the cell surface of Saccharomyces cerevisiae to degrade phytate phosphorus and improve bioethanol production. Appl Microbiol Biotechnol. 2016 Mar;100(5):2449-58. doi: 10.1007/s00253-015-7170-4.
  2. Navone L, Vogl T, Luangthongkam P, Blinco JA, Luna-Flores C, Chen X, von Hellens J, Speight R. Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris. Microb Cell Fact. 2021 Jan 7;20(1):8.


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 593
    Illegal BamHI site found at 1
    Illegal XhoI site found at 925
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 76
    Illegal AgeI site found at 3282
    Illegal AgeI site found at 4157
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3689
    Illegal SapI site found at 987
    Illegal SapI site found at 4094


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