Without getting into any nasty biochem, the gist of it is that proline is a really weirdly shaped amino acid that’s largely responsible for collagen’s structure as a triple helix. Proline’s weird shape causes amino acid chains to turn, helping form the double helix. When proline is hydroxylated (by our favorite prolyl-4-hydroxylase), an OH group is added to the amino acid. This allows more hydrogen bonds to be formed around proline residues in the collagen, reinforcing the exterior’s hydrophilicity.
I essentially answered this in Kathleen’s question above. In brief, it codes for an enzyme that modifies proline residues in collagen. The exterior proline residues, become more hydrophilic, stabilizing the collagen helix.
Essentially, the tRNA used by soybeans during transcription has a different affinity for specific codons than the tRNA found in humans. i.e. it might be better at binding to a UAU than a UAC to make tyrosine. Twist’s algorithm changes the codon for each amino acid to the one that’s best expressed in soybeans, which should in turn cause more translation of the desired gene!
This is very well done! You talked about how you added a promoter upstream of the gene, and on your figure, you call it golden gate assembly. What is this method, and why was it chosen?
The promoter is actually called gmubi, it’s a ubiquitous promoter found in soybeans that increases gene expression of a plasmid. We inserted the promoter using the Golden gate assembly method. Golden Gate revolves around using type ii restriction enzymes to cut specific sequences in two separate strands of DNA to enable them to join together. It’s a super powerful cloning tool that can let you build large gene constructs with a few basic building blocks.
After translation, some parts of the peptide are used to help determine where in the cell it belongs. Our signal sequence flags down a localization protein that lets other machinery in the cell know that our specific product should be exported away.
Hey David, great presentation. How exactly does the hydroxylase make the collagen more stable? Do you know what modification it makes to the collagen’s structure in order to make it more stable?
Kathleen asked a similar question, so I’ll be copy and pasting my answer here.
Without getting into any nasty biochem, the gist of it is that proline is a really weirdly shaped amino acid that’s largely responsible for collagen’s structure as a triple helix. Proline’s weird shape causes amino acid chains to turn, helping form the triple helix. When proline is hydroxylated (by our favorite prolyl-4-hydroxylase), an OH group is added to the amino acid. This allows more hydrogen bonds to be formed around proline residues in the collagen, reinforcing the exterior’s hydrophilicity.
curesymposium.org 
In what ways does the prolyl-4-hydroxylase modify collagen specifically? Is it just the shape or does it actually change its composition?
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Without getting into any nasty biochem, the gist of it is that proline is a really weirdly shaped amino acid that’s largely responsible for collagen’s structure as a triple helix. Proline’s weird shape causes amino acid chains to turn, helping form the double helix. When proline is hydroxylated (by our favorite prolyl-4-hydroxylase), an OH group is added to the amino acid. This allows more hydrogen bonds to be formed around proline residues in the collagen, reinforcing the exterior’s hydrophilicity.
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How does the gene make the structure more stable?
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I essentially answered this in Kathleen’s question above. In brief, it codes for an enzyme that modifies proline residues in collagen. The exterior proline residues, become more hydrophilic, stabilizing the collagen helix.
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Amazing job! Could you touch more on how the Twist Biosciences algorithm works?
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Essentially, the tRNA used by soybeans during transcription has a different affinity for specific codons than the tRNA found in humans. i.e. it might be better at binding to a UAU than a UAC to make tyrosine. Twist’s algorithm changes the codon for each amino acid to the one that’s best expressed in soybeans, which should in turn cause more translation of the desired gene!
LikeLike
This is very well done! You talked about how you added a promoter upstream of the gene, and on your figure, you call it golden gate assembly. What is this method, and why was it chosen?
LikeLike
The promoter is actually called gmubi, it’s a ubiquitous promoter found in soybeans that increases gene expression of a plasmid. We inserted the promoter using the Golden gate assembly method. Golden Gate revolves around using type ii restriction enzymes to cut specific sequences in two separate strands of DNA to enable them to join together. It’s a super powerful cloning tool that can let you build large gene constructs with a few basic building blocks.
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Could you provide a little bit more detail about how the added soybean signal sequence leads to the products being localized outside of the cell?
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After translation, some parts of the peptide are used to help determine where in the cell it belongs. Our signal sequence flags down a localization protein that lets other machinery in the cell know that our specific product should be exported away.
LikeLike
Hey David, great presentation. How exactly does the hydroxylase make the collagen more stable? Do you know what modification it makes to the collagen’s structure in order to make it more stable?
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Kathleen asked a similar question, so I’ll be copy and pasting my answer here.
Without getting into any nasty biochem, the gist of it is that proline is a really weirdly shaped amino acid that’s largely responsible for collagen’s structure as a triple helix. Proline’s weird shape causes amino acid chains to turn, helping form the triple helix. When proline is hydroxylated (by our favorite prolyl-4-hydroxylase), an OH group is added to the amino acid. This allows more hydrogen bonds to be formed around proline residues in the collagen, reinforcing the exterior’s hydrophilicity.
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Hey David, great work! What exactly is Golden Gate assembly, and what makes it different from alternative methods? Thanks!
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