Final Project Log - Produce Ovalbumin with Yeast

Ovalbumin - Pichia Pastoris

Summary: This project will engineer Pichia Pastoris to produce chicken ovalbumin, which represents 54% to 58% of the egg white protein by weight. To achieve this, a codon-optimized chicken ovalbumin gene will be integrated into the yeast expression vector pPIC9K, which has an alpha-factor secretion signal. This will enable the yeast to secrete the ovalbumin directly into the media to simplify downstream processing. In addition to the wet lab experiment, we will also use computational methods to map out the metabolic pathway of this engineered strain and use flux balance analysis to recommend changes to optimize titer.

• Design DNA

  • Host Organism / strain: P. Pick Pastoris (in Genspace inventory)

  • Vector / Plasmid backbone: pPIC9K (in Genspace inventory)

    • https://www.novoprolabs.com/vector/V10245
    • Has an alpha-factor secretion signal so protein will be secreted into the media
    • Same as EML. https://docs.google.com/presentation/d/1F8gBKOCd-tA-tRqyIlwU0Y9oW9Q9e9-x-qS4yx4Zdqk/edit?slide=id.g2ec0bf28272_0_0#slide=id.g2ec0bf28272_0_0

  • DNA / Gene / Insert: https://docs.google.com/spreadsheets/d/1cBF-L2Ks15P8_0sowJyN5cSFtndDfTf-d8JB7Djf5hk/edit?usp=sharing (Twist Codon Optimized) (ORDER THIS)

    • Chicken Ovalbumin DNA: https://www.uniprot.org/uniprotkb/P01012/entry

  • Benchling: https://benchling.com/s/seq-9MUkXze8F97gLHzRKAzz?m=slm-V4tSVwnatjW3TqwduPMq

    • Includes pPIC9K backbone with chick_oval inserted
    • Virtual digest with EcoRI and NotI enzymes

    

  • Restriction enzymes: EcoRI-HF (gaattc) and NotI-HF (gcggccgc) (both in genspace inventory)

  • Selection: G418 antibiotic (in Genspace inventory)?

  • Expression Induction: methanol to induce the AOX1 promoter?

  • Notes

    • Twist Codon Optimization — Sequence = chicken ovalbumin sequence from uniprot; organism = pichia pastoris; sites to avoid = EcoRI (our restriction enzyme), NotI (our restriction enzyme), XbaI (also in inventory), SpeI, BamHI, SnaBI, AvrII, XmaJI, XhoI (potential restriction enzymes we could use)
    • Imported pPIC9K backbone to benchling, found the EcoRI (gaattc) and NotI (gcggccgc) sites in the MCS (multiple cloning site).
      • Final MCS should become: ...GAATTC[codon-optimized ovalbumin]TAAGCGGCCGC…
        • I should add TAA as a stop codon to terminate translation right before the NotI site

  • Questions

    • What else do we need?
      • Do we have media?
      • Transformation reagents? Expression induction?
    • Verify that we have the restriction enzymes EcoRI and NotI
    • How can I tell that the plasmid will ligate / do they have compatible sticky ends?
    • Other components needed
      • Miniprep to get dna out of twist’s bacteria? DNA purification kit?
      • Gibson assembly for construct assembly?
      • Electroporation for transformation of pichia pastoris? Do we need to buy anything for it?
        • Sorbitol?
      • Verify Engineered Strain?
        • G418 antibiotic?
        • Gel electrophoresis?
      • Analyze Protein Expression?
        • Methanol for induction? Expression media?
        • SDS-PAGE Gels? Western blot?
        • Protein purification? Nickel columns — should I add a His-tag in the DNA?

Timeline

• 4/14 — Deadline to send to Twist
• In Between
  • Dry Lab
    • Map out metabolism
    • Flux Balance Analysis
  • Wet Lab
    • Miniprep
    • Gibson Assembly
    • Electroporation
    • Verify engineered strain
    • Analyze protein expression
  • Presentation prep
• 5/14 — Final project presentation

Wet Lab Timeline

• Week 0 (4/15)
  • Become an individual member
  • Order gene fragment twist — restriction site + gene sequence + histag + stop codon
    • Order another fragment without the histag
  • Practice transformation with a pglo
• Week 1 (4/22) — Molecular Cloning
  • Digestion ligation to make the plasmid and verify it
  • Transform pPIC (and make sure pPIC working). Transform it on my own, if possible?
  • Create a glycerol stock on ecoli
• Week 2 (4/29) — Pichia Transformation
  • Transform and grow pichia (could take days to grow)
  • Make Pichia media
• Week 3 (5/6) — Expression induction
  • Methanol induction for AOX1 promoter (Serena could help, check if she has time)