Week 1 HW: Final Project Ideas

Project ZP Freeze

(1) What you would like to biomanufacture and why? I would like to biomanufacture freezing responsive antifreeze proteins like Fr10 and Li16. I am interested in their use in helping to cryopreserve frogs, as well as their (or similar material) potential in helping to preserve animal tissues and organs. As a first stage, I merely wish to create more of them and similar. Source: Biggar KK, Kotani E, Furusawa T, Storey KB. Expression of freeze-responsive proteins, Fr10 and Li16, from freeze-tolerant frogs enhances freezing survival of BmN insect cells. FASEB J. 2013 Aug;27(8):3376-83. doi: 10.1096/fj.13-230573. Epub 2013 May 8. PMID: 23657819. https://pubmed.ncbi.nlm.nih.gov/23657819/ Cai, Q. and Storey, K.B. (1997) ‘Upregulation of a novel gene by freezing exposure in the freeze-tolerant wood frog (Rana sylvatica)’, Gene, 198(1–2), pp. 305–312. Available at: https://doi.org/10.1016/S0378-1119(97)00332-6. McNally, J.D., Wu, S.B., Sturgeon, C.M. and Storey, K.B. (2002) ‘Identification and characterization of a novel freezing-inducible gene, li16, in the wood frog Rana sylvatica’, The FASEB Journal, 16(8), pp. 902–904. Available at: https://doi.org/10.1096/fj.02-0017fje. Le Tri, D., Childers, C.L., Adam, M.K., Ben, R.N., Storey, K.B. and Biggar, K.K. (2019) ‘Characterization of ice recrystallization inhibition activity in the novel freeze-responsive protein Fr10 from freeze-tolerant wood frogs, Rana sylvatica’, Journal of Thermal Biology, 84, pp. 426–430. Available at: https://doi.org/10.1016/j.jtherbio.2019.07.030.

(2) What living system would you use? I would use a microbial system to produce my target, similar to how I produced these proteins via Project Z Freeze from HTBAA in the Spring of 2026. There’s already a wealth of knowledge towards use of microbial systems and prior work lists What’s required to go in would be a specified DNa sequence that encodes the target protein, and an expression plasmid would-d be used for this. Cells carrying this plasmid would receive media that supports their life functions. Outside of the bacteria growing and producing copies of said DNA, transcription would be be used to produce RNA from the tagert gene, followed by translation of the RNA into amino acids that could be further processed to produce to the anti-freeze proteins. The product would be recoeved through mechanical processes that break up the cell and separate the proteins from the rest of the cellular material. The output would hopefully be desired proteins in a defined, stabilizing buffer.

(3) How would you use automation? One automation technology that you would combine with your living system would be automated liquid handling, per tools like an Opentrons. I would appreciate the automation most to reduce variation in products produced through human error and save time. From there, automated screening is a given.

(4) How much would you need to make, and how long would it take? I do not know how much I would need, but I am fine with assuming a small batch and small set of labs. I’d estimate about a month, depending on the facilities available and quality of initial runs. (a) How much? A scale relevant for my project would be 10 labs, giving each 25 mg of purified Fr10 for a total of ~250 mg. Quick calc ~25 mg x 10 labs = ~250 mg.

(b) How long? For the production, it may follow based on this timeline: Preparation of cultures may take 1-4 days. Successful Expression and harvesting could take another 1-4 days. Purification to optimal values could take a solid week: 7 days. Nailing the entire process to a reasonable quality could take another 7 days. Preparing batches, testing shipping, and getting the packaging right could be worth another 2-5 days.
This timing puts us at about 18-27 days. I’m more comfortable saying a month to account for detours.

5. What don’t you know yet? 2-3 of the biggest technical unknowns might be, after answering if the product works in other physical systems tested:

• Have we produced enough for our target applications?
• What forms of the protein are best to construct?
• What are intentional products that could be made along the way to be wary about? After completing your analysis, identify the 2–3 biggest technical unknowns, bottlenecks, or assumptions in your proposed biomanufacturing system. What would you need to learn, measure, or experimentally determine to know whether your idea could actually work?