Week 1 HW: Final Project Ideas

What would you biomanufacture and why?

With a background in industrial design and technology, I want to develop a family of five products made from bacterial cellulose, inspired by the desert. Each product is meant for a different room of a conventional home: the living room, bedroom, kitchen, bathroom, and entry.The main design direction is to let each product grow into a form that is shaped beforehand by a mold. The design language is the desert: dry, mineral, and matte, with a surface that responds to moisture. My research question, from an industrial design perspective, is: can a living material be specified and designed the way we design conventional materials, with a predictable form, finish, and behavior, while still growing as part of the design process? This project is part of my MSc research on Emergence. Here, the final form is not manufactured directly. It emerges from constraints, such as the mold shape and the growth conditions. I have a brief hypothesis on how this project could work. I want to experiment by fabricating 5 identical pieces and placing them in exposure to five different rooms with different humidity levels, which by my hypothesis should generate different designs based on the conditions of which the growth and form are to be made.

What living system would you use?

I plan to use mycelium as the living system for construction. The root network of fungus could spread through the base material and bind into a solid piece that takes the shape of the original mold.Here the variety would be the exposure to heat, once the network is dried the growth stops.

How would you use automation?

I would use automation to make the master forms. Each piece is designed digitally and 3D printed as a master. I then cast the master in silicone to make a mold, and the object grows inside that mold. This gives precise, repeatable molds, so all five pieces share one design language.The rest is done by hand: preparing and sterilizing the growth material, inoculating it, monitoring the growth, removing the piece from the mold, and drying it.

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

I would make five pieces, one per room, each about 20 x 20 x 5 cm. That is 2 L per piece and about 10 L of mold volume in total. I would prepare about 15 L of growth material, to allow for losses and a few test pieces. At an estimated dried density of 0.1 to 0.2 g/cm³, this gives about 1 to 2 kg of finished material. This would take a total of estimated 6 to 9 weeks of work.

Week 2 HW: DNA Design

A secreted hydrophobin, HFBII, as a “moisture dial” for my desert mycelium product family.

AI assistance: Claude (Anthropic). My prompts are listed at the end. In Homework #1 my intro line says “bacterial cellulose”, but the living system I proposed, and the one designed for here, is mycelium.

Part 0Set up in Kernel

My work is in the Kernel repository VazquezFranco-DesertMycelium, in the folder of the same name.

Part 1Define your expression target

AVCPTGLFSNPLCCATNVLDLIGVDCKTPTIAVDTGAIFQAHCASKGSKPLCCVAPVADQALLCQKAIGTF

Part 2Design your genetic parts

Designed DNA (one common codon per amino acid, back-translated by hand):

sp-hfb2 (45 bp)
ATGCAGTTCTTCGCCGTCGCCCTCTTCGCCACCTCCGCCCTCGCC
hfb2-mature (219 bp, includes TGATAA stop)
GCCGTCTGCCCCACCGGCCTCTTCTCCAACCCCCTCTGCTGCGCCACCAACGTCCTCGACCTCATCGGCGTCGACTGCAAGACCCCCACCATCGCCGTCGACACCGGCGCCATCTTCCAGGCCCACTGCGCCTCCAAGGGCTCCAAGCCCCTCTGCTGCGTCGCCCCCGTCGCCGACCAGGCCCTCCTCTGCCAGAAGGCCATCGGCACCTTCTGATAA

Part 3Assemble your construct

A single fungal expression cassette in a reused backbone. The signal peptide and mature sequence share one reading frame and encode the 86-amino-acid precursor, so the protein is made and then secreted. The coding region is 264 bp (45 + 213 + 6 for the TGATAA stop) and reads MQFFAVALFATSALA straight into AVCPTGLFSN….

Part 4Optimize and quality-check

Codon optimization

Codon usage matters because a host’s tRNA pools favor some codons, so a gene written with rarely used codons can be translated slowly or inaccurately. My draft already uses common, GC-ending codons.

Sequence search

Checks whether my sequence matches known genes. It encodes a natural protein through a designed DNA sequence, so I compare the protein-level and DNA-level matches separately.

Safety screen

Screens the construct against databases of sequences of concern.

Part 5From design to build

How would I build it?

The gene is small (264 bp), but the backbone and regulatory parts are much longer. I would order the coding region as synthetic DNA with overlaps to its neighbors and join it to the regulatory parts and backbone by Gibson assembly⁵, where overlapping fragments are joined in one isothermal reaction by an exonuclease, a polymerase and a ligase. A chip-synthesized oligo pool pays off when many genes or variants are built together^6,8; for one gene it is not worth it.

What does synthesis constrain?

Synthesis has error rates that grow with length and complexity. Error correction such as the mismatch-binding approach of Carr et al.⁷ removes faulty molecules, and I would sequence-verify the final construct. My design has two trouble spots. My back-translation is about 69% GC (calculated from the designed sequence), because I picked C/G-ending codons, and very high GC makes synthesis and assembly harder. The paired cysteines also create short repeats such as TGCTGC. Shorter fragments of moderate GC joined by overlaps are easier to make and can each be checked before assembly.

Prototype vs. scale

The prototype is one construct built once: a synthetic coding fragment, Gibson assembly, sequencing, then fungal transformation. At product scale DNA is not used up: once a strain carries the cassette, the fungus copies it as it grows, so five 20 × 20 × 5 cm pieces (about 15 L of growth material) come from growing mycelium, not from making more DNA. DNA synthesis scale matters for design exploration. To tune moisture behavior I would want several expression levels (promoter and UTR variants), and there a pooled oligo approach⁶ becomes attractive.

Part 6What don’t I know yet?

ReferencesAll references on this page

1. UniProt entry P79073, Hydrophobin-2, Trichoderma reesei. uniprot.org/uniprotkb/P79073
2. Nakari-Setälä, T. et al. (1997). Differential expression of the vegetative and spore-bound hydrophobins of Trichoderma reesei: cloning and characterization of the hfb2 gene. Eur. J. Biochem. 248(2), 415–423.
3. Hakanpää, J. et al. (2004). Atomic resolution structure of the HFBII hydrophobin, a self-assembling amphiphile. J. Biol. Chem. 279, 534–539.
4. Kottmeier, K., Ostermann, K. & Rödel, G. Secretion system on the basis of a hydrophobin signal sequence from Trichoderma reesei. Patent WO2011073367A1 (2011). Source of the 15-residue HFBII leader (SEQ ID No. 14).
5. Gibson, D. G. et al. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6, 343–345. 10.1038/nmeth.1318
6. Kosuri, S. et al. (2010). Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nature Biotechnology 28, 1295–1299. 10.1038/nbt.1716
7. Carr, P. A. et al. (2004). Protein-mediated error correction for de novo DNA synthesis. Nucleic Acids Research 32, e162. 10.1093/nar/gnh160
8. Tian, J. et al. (2004). Accurate multiplex gene synthesis from programmable DNA microchips. Nature 432, 1050–1054. 10.1038/nature03151

Final Project