Week 12

Node participant note: I am a remote Genspace node listener based in Nigeria without onsite lab access. The Week 12 lab was a bioproduction session at Genspace nodes. I engaged with the Building Genomes lecture content fully and document that engagement below. The ÌṢỌ project constraints memo is included as the project deliverable for this week.

Class Assignment — Week 12

Part A. Building Genomes: Course Notes

Core Themes from the Week 12 Lectures

The Building Genomes week brings together two convergent threads: the technical capacity to synthesise and assemble DNA at genome scale, and the design question of what you would build if synthesis cost were not a constraint.

Prof Church’s framing of genome-scale engineering through GP-write (Genome Project-write) positioned this not just as a sequencing problem in reverse, but as a design problem with hard biosafety constraints built into the architecture. The recoded organism work from his group (the 57-codon E. coli described by Fredens et al., 2019) demonstrated that synonymous codon compression is technically feasible at genome scale and creates a substrate for radical biocontainment: a cell whose codon table is incompatible with natural horizontal gene transfer cannot receive functional genes from wild-type organisms, and cannot donate them in return. That is a containment approach that operates at the informational layer rather than the metabolic layer.

The Glass/JCVI approach from the Mycoplasma mycoides JCVI-syn3.0 work brought a different emphasis: minimum genome definition. Synthesising a 531-gene essential genome and systematically knocking out non-essential genes revealed that roughly a third of essential gene functions are genuinely unknown in the minimal cell. That is a striking statement about the limits of our functional annotation of even the simplest known organisms.

Prof Boeke’s work on Sc2.0 (synthetic yeast genome) showed what large-scale genome synthesis looks like in a eukaryotic system: chromosome-by-chromosome replacement, with SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution) built in as a built-in evolutionary exploration tool. The loxP site insertion throughout the synthetic chromosomes is a design choice that converts the genome into a substrate for combinatorial rearrangement on demand.

Connection to ÌṢỌ Containment Architecture

The containment architecture in ÌṢỌ is currently a first-generation metabolic dependency (ΔdapA auxotrophy, requiring exogenous DAP for survival). This is the conventional approach and it works at the population level, but it has a known failure mode: reversion or suppressor mutations can restore DAP synthesis at low frequency over generational time, and horizontal acquisition of a wild-type dapA gene from environmental bacteria remains theoretically possible.

The recoded organism approach points toward a second-generation containment strategy that would complement rather than replace auxotrophy: if ÌṢỌ’s key functional genes were encoded using a compressed codon table incompatible with natural ribosomes, horizontal gene transfer from or to wild-type organisms would be informationally blocked. This is a long-term design goal rather than a Spring 2026 deliverable, but the GP-write literature makes the design path concrete.

Part B. Project Constraints Memo: ÌṢỌ Design Boundaries (Spring 2026)

What ÌṢỌ is

A model-first, constraint-aware computational framework for engineering E. coli Nissle 1917 as a gut sentinel. The project produces reproducible computational models, tradeoff analyses (fitness vs efficacy), robustness assessments, and design regime maps. The current deliverable is a set of ODE and evolutionary models that inform what to build, not a built organism.

Design constraints actively governing current choices

Fitness budget: Every functional addition (biosensor, effector, containment circuit) carries a metabolic cost. The ODE model tracks growth rate as an explicit variable. No module is added without a corresponding fitness penalty estimate. The project is designed around stable, low-burden expression rather than peak performance.

Selection pressure: The model assumes selection is always running. Any design that is only stable at the intended expression level but unstable under evolutionary pressure is treated as a failed design, not a promising candidate awaiting optimization.

Containment as a first-class design variable: ΔdapA auxotrophy is included not as an afterthought but as a parameter in the escape probability model. The Luria-Delbrück framework used to estimate reversion frequency treats containment failure as a quantifiable risk to be designed against, not a worst-case scenario to be hoped away.

Ecological realism: The gut is not a flask. The models include a competing commensal term and treat the ÌṢỌ organism as one species in a dynamic ecosystem, not a cell culture in isolation.

What is out of scope (Spring 2026)

  • Wet-lab validation of any construct
  • Full microbiome ecosystem simulation
  • Regulatory pathway analysis
  • Clinical or preclinical deployment planning
  • Any in vivo animal model work

Next steps beyond Spring 2026

The Twist construct (MccH47_pUC19_EcN_v1) is the bridge to Phase 2. If synthesis is confirmed and the sequence is validated, in vitro characterisation of TtrR-mediated induction and MccH47 expression can proceed in a collaborating laboratory environment. Cloud lab platforms would be the preferred route given my remote location.

Works Cited

Fredens, J., Wang, K., de la Torre, D., Funke, L. F. H., Robertson, W. E., Christova, Y., Chia, T., Schmied, W. H., Dunkelmann, D. L., Beránek, V., Uttamapinant, C., Llamazares, A. G., Elliott, T. S., & Chin, J. W. (2019). Total synthesis of Escherichia coli with a recoded genome. Nature, 569(7757), 514–518. https://doi.org/10.1038/s41586-019-1192-5

Hutchison, C. A., Chuang, R.-Y., Noskov, V. N., Assad-Garcia, N., Deerinck, T. J., Ellisman, M. H., Gill, J., Kannan, K., Karas, B. J., Ma, L., Pelletier, J. F., Qi, Z.-Q., Richter, R. A., Strychalski, E. A., Sun, L., Suzuki, Y., Tsvetanova, B., Wise, K. S., Smith, H. O., … Glass, J. I. (2016). Design and synthesis of a minimal bacterial genome. Science, 351(6280), aad6253. https://doi.org/10.1126/science.aad6253

Richardson, S. M., Mitchell, L. A., Stracquadanio, G., Yang, K., Dymond, J. S., DiCarlo, J. E., Lee, D., Huang, C. L., Chandrasegaran, S., Cai, Y., Boeke, J. D., & Bader, J. S. (2017). Design of a synthetic yeast genome. Science, 355(6329), 1040–1044. https://doi.org/10.1126/science.aaf4557

Lajoie, M. J., Rovner, A. J., Goodman, D. B., Aerni, H.-R., Haimovich, A. D., Kuznetsov, G., Mercer, J. A., Wang, H. H., Carr, P. A., Mosberg, J. A., Rohland, N., Schultz, P. G., Jacobson, J. M., Rinehart, J., Church, G. M., & Isaacs, F. J. (2013). Genomically recoded organisms expand biological functions. Science, 342(6156), 357–360. https://doi.org/10.1126/science.1241459

AI Prompts Employed (Claude AI)

  • Summarise the key design principles of GP-write and how recoded organisms differ from standard auxotrophic containment strategies
  • Explain how SCRaMbLE works in the Sc2.0 synthetic yeast and what it reveals about genome architecture
  • What is the minimum genome concept from JCVI-syn3.0 and what fraction of essential gene functions remain unknown
  • Connect recoded organism containment logic to the ÌṢỌ ΔdapA auxotrophy as complementary rather than competing approaches