Week 1 HW: Biomaterial of PVA and PEG funtionalized with ECM proteins for burn wound healing

(1) What you would like to biomanufacture and why?

I would like to manufacture biomaterials for burns. I see this as a big problem because it has cost too many lives. In countries like Ecuador, burns do not have a first aid product that could help reduce the damage that could be caused by the remaining heat. For this reason, I would like to construct a biomaterial that could hold bioactives from fruits and growth factors to reduce inflammation and remaining damage. I consider this an important issue abroad because in some homes there are children who could be burned unintentionally because they are playing or exploring. I do not want to find guilt at home; I want to give parents support in case their children get burned. I would like to program fibroblasts to produce a thin film rich in extracellular proteins such as collagen, fibronectin, laminin, and gelatin, and Fibroblast Growth Factors to replace the burned proteins, to be loaded in a hydrogel matrix. These proteins are essential for the development of first aid for burns because they can be dried. Therefore, it can be a dried product that just needs water to act and could be preserved for a long time.

(2) What living system would you use?

I would like to produce growth factors and extracellular matrix proteins from modified fibroblasts to be held in a hydrogel matrix with banana bioactives that calm the wound. My proposal is the optimized production of fibroblasts in a 2D culture in order to generate deposits of these proteins. Then these cultures should be decellularized to obtain just the proteins. Those will then be dried by freeze-drying to be incorporated in the hydrogel. The hydrogel will be formulated with PVA, PEG, and functionalized with banana bioactives. Once the hydrogel is formulated with ECM proteins, matrix, and bioactives, it will be dried to prolong its durability.

(3) How would you use automation?

I’d automatize the section of ECM protein production because I could reduce the contamination and tissue died by human mistakes. I also automatize the hydrogel formulation because I could standardize the quality and consistency of fiscal and chemical properties of hydrogel.

(4) How much would you need to make, and how long would it take?

Perform a back-of-the-envelope calculation estimating what it would take to manufacture your target at a meaningful scale.

(a) How much?

For this question, I ask help from gemini ai because I was not sure how to make it. I explained my product and that my goal is to reach 1,000 burn patients who will receive a hydrogel containing 0.5% banana bioactives, 1% ECM proteins, 3.25% PVA, and 5% PEG. Therefore, taking in count this assumptions: Average burn wound surface area per patient: 100 cm2 (approx. 10 cm x 10 cm dressing per patient). Thickness of the dry hydrogel dressing: 2 mm (0.2 cm). Hydrogel density: approx. 1 g/cm3. Hydrogel formulation (% w/w of total dry hydrogel weight): PEG: 5.0% PVA: 3.25% ECM proteins: 1.0% Banana bioactives: 0.5% The approximated component breakdown of is the following: Volume per dressing: Volume = 100 cm2 x 0.2 cm = 20 cm3 per dressing Mass per dressing: Mass = 20 cm3 x 1 g/cm3 = 20 g of hydrogel matrix per patient Total annual production (for 1,000 patients): Total Hydrogel Matrix = 1,000 x 20 g = 20,000 g = 20 kg/year Component breakdown per year (20 kg total): PEG (5.0%): 20 kg x 0.050 = 1.0 kg/year PVA (3.25%): 20 kg x 0.0325 = 0.65 kg (650 g)/year Required ECM proteins (1.0%): 20 kg x 0.010 = 0.20 kg (200 g)/year Required Banana bioactives (0.5%): 20 kg x 0.005 = 0.10 kg (100 g)/year

(b) How long?

Continuing with the same production, I use Gemini to help me to resolve. So, the production parameters and assumptions are: ECM Protein Yield: Modified fibroblasts in 2D culture yield approximately 10 µg of ECM proteins per cm2 per 2-week culture cycle. 2D Culture Area Needed: To yield 200 g (200,000,000 µg) of ECM proteins per year: Total Area = 200,000,000 µg / 10 µg/cm2 = 20,000,000 cm2 = 2,000 m2 of total culture area per year. Batch Cycle Time: 14 days for cell growth + 2 days for decellularization, freeze-drying, and hydrogel mixing = 16 days per batch cycle (approx. 22 batches per year). Then, the automation and process timeline are: Prototyping vs. Scale: At prototype scale, 2D cell culture, decellularization, bioactive extraction, and hydrogel formulation are performed manually in standard T-flasks and small batches. To scale up to produce 20 kg of matrix (including 200 g of ECM, 100 g of banana bioactives, 1 kg of PEG, and 650 g of PVA), manual processing is too slow and variable. Automated Solution: We utilize an automated high-throughput liquid handling system (e.g., robotic arms for media changes, automated rinsing for decellularization, and automated freeze-dryers) running multi-layer cell factories (6,320 cm2 per tray). Production Rate: Operating 15 multi-layer cell factory stacks in parallel, each 16-day cycle produces approx. 9.1 g of ECM proteins, enough for 910 g of complete hydrogel formulation (~45 dressings per batch). Total Time to Target: Manufacturing the target quantity of 20 kg of biomaterial (1,000 treatments/year) would take approximately 352 days (22 consecutive batch cycles), operating continuously across the year.

(5) What don’t you know yet?

I think the unknown things I need to learn are first the cell production automatization, and the correct estimation of cost and yield of the project.

Week 2 HW: DNA Design

Project Overview

For this project, I want to potency my current hydrogel for burn wound healing by adding it extracellular matrix proteins (ECM) and growth factors (GF). So, following this idea, the DNA design will focus on the production of the ECM and GF.

Kernel repository

HTBAA_HW2_Geanella Flores

Part 1: Define your expression target

What molecule are you expressing?

The target to express will be the Epidermal Grow Factor, Insuline-like Grow Factor and the Extracellular Matrix.

What is the sequence?

EGF_HUMAN

MLLTLIILLPVVSKFSFVSLSAPQHWSCPEGTLAGNGNSTCVGPAPFLIFSHGNSIFRIDTEGTNYEQLVVDAGVSVIMDFHYNEKRIYWVDLERQLLQRVFLNGSRQERVCNIEKNVSGMAINWINEEVIWSNQQEGIITVTDMKGNNSHILLSALKYPANVAVDPVERFIFWSSEVAGSLYRADLDGVGVKALLETSEKITAVSLDVLDKRLFWIQYNREGSNSLICSCDYDGGSVHISKHPTQHNLFAMSLFGDRIFYSTWKMKTIWIANKHTGKDMVRINLHSSFVPLGELKVVHPLAQPKAEDDTWEPEQKLCKLRKGNCSSTVCGQDLQSHLCMCAEGYALSRDRKYCEDVNECAFWNHGCTLGCKNTPGSYYCTCPVGFVLLPDGKRCHQLVSCPRNVSECSHDCVLTSEGPLCFCPEGSVLERDGKTCSGCSSPDNGGCSQLCVPLSPVSWECDCFPGYDLQLDEKSCAASGPQPFLLFANSQDIRHMHFDGTDYGTLLSQQMGMVYALDHDPVENKIYFAHTALKWIERANMDGSQRERLIEEGVDVPEGLAVDWIGRRFYWTDRGKSLIGRSDLNGKRSKIITKENISQPRGIAVHPMAKRLFWTDTGINPRIESSSLQGLGRLVIASSDLIWPSGITIDFLTDKLYWCDAKQSVIEMANLDGSKRRRLTQNDVGHPFAVAVFEDYVWFSDWAMPSVMRVNKRTGKDRVRLQGSMLKPSSLVVVHPLAKPGADPCLYQNGGCEHICKKRLGTAWCSCREGFMKASDGKTCLALDGHQLLAGGEVDLKNQVTPLDILSKTRVSEDNITESQHMLVAEIMVSDQDDCAPVGCSMYARCISEGEDATCQCLKGFAGDGKLCSDIDECEMGVPVCPPASSKCINTEGGYVCRCSEGYQGDGIHCLDIDECQLGEHSCGENASCTNTEGGYTCMCAGRLSEPGLICPDSTPPPHLREDDHHYSVRNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRHAGHGQQQKVIVVAVCVVVLVMLLLLSLWGAHYYRTQKLLSKNPKNPYEESSRDVRSRRPADTEDGMSSCPQPWFVVIKEHQDLKNGGQPVAGEDGQAADGSMQPTSWRQEPQLCGMGTEQGCWIPVSSDKGSCPQVMERSFHMPSYGTQTLEGGVEKPHSLLSANPLWQQRALDPPHQMELTQ

IGF1_HUMAN

MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKYQPPSTNKNTKSQRRKGWPKTHPGGEQKEGTEASLQIRGKKKEQRREIGSRNAECRGKKGK

ECM2_HUMAN

MKIAVLFCFFLLIIFQTDFGKNEEIPRKQRRKIYHRRLRKSSTSHKHRSNRQLGIQQTTVFTPVARLPIVNFDYSMEEKFESFSSFPGVESSYNVLPGKKGHCLVKGITMYNKAVWSPEPCTTCLCSDGRVLCDETMCHPQRCPQTVIPEGECCPVCSATVSYSLLSGIALNDRNEFSGDSSEQREPTNLLHKQLPPPQVGMDRIVRKEALQSEEDEEVKEEDTEQKRETPESRNQGQLYSEGDSRGGDRKQRPGEERRLAHQQQRQGREEEEDEEEEGEEGEEDEEDEEDPVRGDMFRMPSRSPLPAPPRGTLRLPSGCSLSYRTISCINAMLTQIPPLTAPQITSLELTGNSIASIPDEAFNGLPNLERLDLSKNNITSSGIGPKAFKLLKKLMRLNMDGNNLIQIPSQLPSTLEELKVNENNLQAIDEESLSDLNQLVTLELEGNNLSEANVNPLAFKPLKSLAYLRLGKNKFRIIPQGLPGSIEELYLENNQIEEITEICFNHTRKINVIVLRYNKIEENRIAPLAWINQENLESIDLSYNKLYHVPSYLPKSLLHLVLLGNQIERIPGYVFGHMEPGLEYLYLSFNKLADDGMDRVSFYGAYHSLRELFLDHNDLKSIPPGIQEMKALHFLRLNNNKIRNILPEEICNAEEDDDSNLEHLHLENNYIKIREIPSYTFSCIRSYSSIVLKPQNIK

What host or chassis?

Mammalian fibroblast line

Where does the production need to go?

The proteins produced need to be secreted outside of the cell.

Part 3: Assemble your construct

Part 4: Optimize and quality-check

Week 3 HW: Cell-Free Protocols

CFPS REACTION DESIGN FOR SFGFP, PETASE, RETEPLASE

GOAL: using the collective intelligence of the HTBAA community and accessible AI systems, design novel CFPS reaction compositions for each of the three target proteins: sfGFP, PETase, and Reteplase to reduce the cost per gram of functional protein. Each design should be compatible with the experimental framework described above and should reflect the distinct expression and folding requirements of that protein. You can learn more about the details of the coopetition.

COMPARE CELL-FREE REACTION COMPOSITIONS (20 minutes)

Final Project