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MCDB 3000: Engineering the Soybean to Produce Squalene and Bovine Milk

Spring 2022

Lindsey Neukirch & Charlotte Sprason

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Why Make Bovine Milk in Soybeans?

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The Casein Micelle Construct

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Experimental Design of Casein Gene Construct Insertion

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How Golden Gate Works

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Casein Restriction Digest Results

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What is Squalene?

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Squalene and the Plant Sterol Biosynthesis Pathway

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  • Squalene is a pathway intermediate

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  • Increase squalene levels by:
    • Upregulating synthesis
    • Inhibit downstream metabolism

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  • Achieved by:
    • Increasing Squalene Synthase copy number
    • Knocking down Squalene Epoxidase mRNA by RNAi

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  • 7 paralogs squalene epoxidase

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  • ~200bp sequence target fragment from different regions each paralog
    • Determined by multiple sequence alignment

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Designing an RNAi Construct

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Assembling Plasmid Vectors

  • Type 2 Restriction enzymes used cleave both destination vector and insert(s)

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  • Selection by antibiotic resistance and blue/white

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  • Alternate antibiotic resistances destination vector

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  • Constitutive promoter and terminator

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What Did we Make?

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Restriction Digest Plasmid Verification

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Going Industrial

  1. Insert construct with GFP into Agrobacterium

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  1. Transform Soybean cells and observe fluorescence

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  1. Isolate and test for desired product(s) from successfully transformed regions

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  1. Rebuild construct with inducible promoter

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  1. Attempt to transform cells become germline therefore create fully hybrid plant