Difference between revisions of "Part:BBa K912001"
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    <title>Contribution: BBa_K912001 (appa phytase)</title>
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    <h1>Contribution: BBa_K912001 (appa phytase)</h1>
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    <h2>Summary</h2>
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    <p>We have made contributions on the original components BBa_K912001. Throughout the design, we used GAL1 promoters (BBa_K4870008) to induce protein expression using galactose and added MF-alpha-1(BBa_K4870009), which is N-terminal secretion signal from S. cerevisiae alpha-factor, and connected SED1 gene (BBa_K4870011) through G4S 3-linker (BBa_K4870010). Fusion expression of phytase encoding genes AppA with anchoring protein encoding genes SED1 on the cell surface to produce yeast single cell proteins with phytase activity, which can be added to feed to enhance animal nutrition and absorption.</p>
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    <h2>Documentation</h2>
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    <h3>Usage and Biology</h3>
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    <p>Appa (appa phytase) acts to break down phytic acid into inositol, releasing chelated iron and other micronutrients. Phytase belongs to the histidine or alkaline phosphatase family and can catalyze the hydrolysis of phytic acid to gradually produce inositol derivatives and inorganic phosphates. Therefore, phytase is usually used as a commercial feed additive to improve the digestibility of phytic acid in grain raw materials. On the one hand, it increases the animal's absorption of phosphate in feed, and on the other hand, it reduces the anti nutritional effect of phytic acid.</p>
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    <h3>Characterization/Measurement</h3>
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    <p>To construct the plasmid pYES2-Hyg-GAL1p-α-AppA-SED1-RPL41Bt, we used the E. coli DH5α genomic DNA as a template to amplify the AppA sequence by PCR. Then, the AppA sequence was inserted into the SphⅠ and AvrⅡ sites of plasmid pYES2-Hyg-GAL1p-α-SED1-RPL41Bt, by restriction endonuclease digestion and linkage.</p>
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    <p>The plasmid was then transferred into the E. coli DH5α competent cells. The result showed that transformants were successfully grown after overnight culture. After colony PCR verification, the recombinants had the expected bands (1293 bp), indicating the successful transformation of this plasmid.</p>
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    <figure>
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        <img src="https://static.igem.wiki/teams/4870/wiki/bba-k912001/2.png" alt="Figure 2" width="500">
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        <figcaption>Figure 2: Construction of the AppA display plasmid</figcaption>
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    </figure>
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    <h2>References</h2>
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    <ol>
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        <li>Dersjant-Li Y, Bello A, Stormink T, Abdollahi MR, Ravindran V, Babatunde OO, Adeola O, Toghyani M, Liu SY, Selle PH, Marchal L. Modeling improvements in ileal digestible amino acids by a novel consensus bacterial 6-phytase variant in broilers. Poult Sci. 2022 Mar;101(3):101666.</li>
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        <li>Acquistapace IM, Thompson EJ, Kühn I, Bedford MR, Brearley CA, Hemmings AM. Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase, AppA. Int J Mol Sci. 2022 Jun 6;23(11):6346.</li>
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        <li>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. doi: 10.1186/s12934-020-01499-7.</li>
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Revision as of 09:00, 10 October 2023


E. coli phytase (appa phytase)

Part to produce the appa e. coli phytase. This phytase acts to break down phytic acid into inositol, releasing chelated iron and other micronutrients.


HokkaidoU_Japan 2017 analyzed this part. This protein's activity falls as below graph.

Protocol: 50μl 7.5 mM Phytic acid 190μl CH3COOH Buffer

Digest phytic acid in above situation with sample phytase solution 10μl ,37degrees celcius, 10 min.

After digestion, using AMM solution (2ml of 10mM NH4Mo7O2.4H2O: 5N H2SO4: Acetone= 1:1:2) analyze Optical Absorbance. The more Pi are there, the more yellow AMM becomes.

T--HokkaidoU_Japan--K912001gurafu.png

<!DOCTYPE html> Contribution: BBa_K912001 (appa phytase)

Contribution: BBa_K912001 (appa phytase)

Summary

We have made contributions on the original components BBa_K912001. Throughout the design, we used GAL1 promoters (BBa_K4870008) to induce protein expression using galactose and added MF-alpha-1(BBa_K4870009), which is N-terminal secretion signal from S. cerevisiae alpha-factor, and connected SED1 gene (BBa_K4870011) through G4S 3-linker (BBa_K4870010). Fusion expression of phytase encoding genes AppA with anchoring protein encoding genes SED1 on the cell surface to produce yeast single cell proteins with phytase activity, which can be added to feed to enhance animal nutrition and absorption.

Documentation

Usage and Biology

Appa (appa phytase) acts to break down phytic acid into inositol, releasing chelated iron and other micronutrients. Phytase belongs to the histidine or alkaline phosphatase family and can catalyze the hydrolysis of phytic acid to gradually produce inositol derivatives and inorganic phosphates. Therefore, phytase is usually used as a commercial feed additive to improve the digestibility of phytic acid in grain raw materials. On the one hand, it increases the animal's absorption of phosphate in feed, and on the other hand, it reduces the anti nutritional effect of phytic acid.

Characterization/Measurement

To construct the plasmid pYES2-Hyg-GAL1p-α-AppA-SED1-RPL41Bt, we used the E. coli DH5α genomic DNA as a template to amplify the AppA sequence by PCR. Then, the AppA sequence was inserted into the SphⅠ and AvrⅡ sites of plasmid pYES2-Hyg-GAL1p-α-SED1-RPL41Bt, by restriction endonuclease digestion and linkage.

The plasmid was then transferred into the E. coli DH5α competent cells. The result showed that transformants were successfully grown after overnight culture. After colony PCR verification, the recombinants had the expected bands (1293 bp), indicating the successful transformation of this plasmid.

Figure 2
Figure 2: Construction of the AppA display plasmid

References

  1. Dersjant-Li Y, Bello A, Stormink T, Abdollahi MR, Ravindran V, Babatunde OO, Adeola O, Toghyani M, Liu SY, Selle PH, Marchal L. Modeling improvements in ileal digestible amino acids by a novel consensus bacterial 6-phytase variant in broilers. Poult Sci. 2022 Mar;101(3):101666.
  2. Acquistapace IM, Thompson EJ, Kühn I, Bedford MR, Brearley CA, Hemmings AM. Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase, AppA. Int J Mol Sci. 2022 Jun 6;23(11):6346.
  3. 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. doi: 10.1186/s12934-020-01499-7.


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 AgeI site found at 688
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1213