Week 2 HW: DNA Design

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HTBAA Homework #2 Due: Tuesday September 29, by 2:30p ET

Designing with the objective of optimizing potatoes as a staple food for human habitation (a Mars colony) on Mars.

  1. Rapid urea recovery (DUR3) / Conversion of malodorous compounds into tryptophan (an amino acid)
  2. LED-specific artificial light-harvesting complex (LHC) / Constant release of tuber-formation signals (StSP6A)
  3. Accumulation of heme iron and calcium / Expression of potent antioxidant molecules (astaxanthin)
  4. Complete knockout of the solanine synthesis master gene (SSR2)

① 24-hour non-stop tuber formation circuit

A circuit designed to induce ultra-fast tuber formation regardless of day length, even under continuous 24-hour LED illumination.

●Promoter: p35S (CaMV 35S Promoter) or pNos

●RBS (5’UTR): Tobacco Mosaic Virus Ω (omega) leader

●CDS (Gene): StSP6A (codon-optimized sequence)

●Terminator: tNOS (Nopaline Synthase Terminator)

②: High-speed human urine (urea)/nitrogen recovery circuit

●Promoter: pPatatin (Patatin Promoter / B33)

●RBS (5’UTR): StPatatin 5’UTR

●CDS (Gene): DUR3 (derived from Sinorhizobium or plants)

●Terminator: t35S (CaMV 35S) Terminator)

③: CRISPR cassette for the complete elimination of toxins (solanine)

●Promoter: pU6 (U6/U3 promoter derived from Arabidopsis or potato)

●RBS (5’ UTR): None

●CDS / RNA (gene): gRNA targeting StSSR2

●Terminator: Poly-T (T6: TTTTTT)

④: Circuit for enhancing countermeasures against radiation and low gravity (astaxanthin and calcium)

Multi-cistronic (linked) circuit for accumulating nutrients optimized for humans within the potato

●Promoter: pSuper or pMac (strong hybrid plant promoter)

●RBS (5’ UTR): g10L (leader optimized for plant expression)

●CDS (genes): crtW / crtZ / StCalreticulin (P2A-linked)

●Terminator: tOcs (Octopine Synthase Terminator)

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Focus on ① of the four sets.

① 24-hour non-stop tuber formation circuit

A circuit designed to induce ultra-fast tuber formation regardless of day length, even under continuous 24-hour LED illumination.

●Promoter: p35S (CaMV 35S Promoter) or pNos

Overview: A strong constitutive promoter derived from the Cauliflower Mosaic Virus; it is the most widely used promoter in the plant kingdom, driving continuous transcription 24 hours a day.

●RBS (5’ UTR): Tobacco Mosaic Virus Ω (omega) leader

Overview: The Ω sequence (a translational enhancer) from the Tobacco Mosaic Virus. It strongly recruits ribosomes within plant cells, dramatically boosting translation efficiency.

●CDS (Gene): StSP6A (codon-optimized sequence)

Overview: A gene encoding the tuber-formation signal in potato (Solanum tuberosum). Processing in Kernel: The “Codon Optimization” function is used to optimize the nucleotide sequence based on potato codon usage bias (Target organism: Solanum tuberosum).

●Terminator: tNOS (Nopaline Synthase Terminator)

Overview: A standard plant terminator derived from Agrobacterium. It ensures the reliable termination of transcription and stabilizes the mRNA.

Chassis: Solanum tuberosum (Potato)

Part 1: Define your expression target Start from the project you proposed in Homework #1 and pin down exactly what your cells need to make. Answer in your write-up: ● What molecule are you expressing? A protein, an RNA, or an enzyme (or small set of enzymes) for a pathway. If your HW1 product is a material, food, or chemical, identify the protein(s) that produce it and design for those. ● What is the sequence? Give the protein or RNA sequence, or the specific gene, you will encode, and say where it comes from (a database accession such as UniProt or NCBI, a known part, or de novo). Cite it. ● What host or chassis? The living system from HW1, for example E. coli, S. cerevisiae, CHO or another mammalian line, or a cell-free system. Your host drives almost every part choice that follows. ● Where does the product need to go? Cytoplasm, secreted, membrane, or a compartment? This decides whether you need a signal peptide or targeting sequence.

Part 2: Design your genetic parts Every construct is built from parts. Decide which parts your target needs, then create or import each one in Kernel with the correct part type. At minimum, choose the elements below for your host.

For each part you use, record in Kernel (and note in your write-up):

  1. The part type and a short name.
  2. Its sequence and where it came from (a Kernel part, a database, or designed).
  3. One sentence of justification, why this part, for this host, for this product. You do not need to hand-design a backbone from scratch; you may reuse an existing one and say what it provides.

Part 3: Assemble your construct Now put the parts together into a single construct in Kernel.

  1. Create a new construct in your folder and add your parts in the correct 5’→3’ order for your host (for a basic expression cassette: promoter → RBS or 5’ UTR/Kozak → CDS → terminator, plus any signal peptide or tag, in the vector backbone).
  2. Check the assembled sequence: the reading frame is intact, the start and stop codons are where you expect, and there are no accidental internal stops.
  3. Look at the construct map Kernel renders and confirm the parts sit in the order and orientation you intended. Deliverable for this part: your assembled construct in Kernel, plus an exported schematic of it in your write-up, with a brief description of the design.

Part 4: Optimize and quality-check Use Kernel’s design tools on your coding sequence and construct, and interpret the results rather than just running them. ● Codon-optimize your CDS for your chosen host. Compare before and after: what changed, and why does codon usage matter for expression in this organism? Note any constraints you applied (e.g. avoiding certain restriction sites, GC content). ● Search your sequence (BLAST / sequence search) against known sequences. Is your CDS what you think it is? Is it identical to a natural gene, or novel? What is the closest match? ● Run the safety screen on your construct. Summarize what it checks for and what it reports. If it flags something, explain whether it is expected for your design and how you would respond. Deliverable for this part: a short paragraph per tool covering what you ran, what it returned, and what you concluded. Screenshots welcome.

Part 5: From design to build A sequence in software is not yet DNA. Using this week’s readings, reason about how your construct would actually be made. Answer in your write-up: ● How would you build it? Would you order it as synthetic DNA, assemble it from smaller fragments (e.g. Gibson assembly), or build it from a pool of oligos synthesized on a chip? Justify your choice given your construct’s length and complexity. ● What does synthesis constrain? Synthesis has error rates and struggles with certain sequences. Does your design contain anything hard to synthesize? How would error correction or fragment-based assembly change your approach? ● Prototype vs. scale. Mirroring HW1: the way you’d build one construct to test your idea may differ from how you’d produce DNA at the scale your product needs. Briefly describe both if they differ. Ground this in at least two of the assigned readings and cite them.

Part 6:What don’t you know yet? As in Homework #1, close by naming the edges of your design. Identify the 2–3 biggest unknowns or risks in your construct. For example: Will the promoter give the right expression level? Will the protein fold and be active in this host? Will it be secreted? Is codon optimization enough, or do you need to tune it empirically? For each, state how you would find out: the measurement, experiment, or design variant you would try next. A good answer here shows you know which decisions are still bets.