Week 2 HW: DNA Design

HTBAA Homework #2 — Genetic construct design

Part 0: Set up

Kernel access is still being sorted, so I’ve built this design in Benchling in the meantime.

This it the T7 phage tail redesigned (Chain A and Chain B) with different constructs for each chain.

Kernel repo link: https://kernel.asimov.com/htbaa2026/repositories/repository/4c6c7d60-e7e9-46e2-a114-50b14840ab12/folder/a97c097f-48f2-4aab-b090-4b3a864207c0

Part 1: Expression target

What I’m expressing. A protein: a soluble, engineered receptor-binding domain (RBD) derived from the bacteriophage T7 tail fibre protein gp17, redesigned to bind OprF on Pseudomonas aeruginosa. In the intact phage this domain sits at the tip of the tail fibre and does the work of recognising the host surface; here I’m expressing it on its own, as a binding reagent rather than as part of a virion.

Why this, given my HW1 proposal. My HW1 project was a platform matching bioproducts and biologies to bioresidue feedstocks. Residue-fed fermentations are among the least sterile inputs in industrial biomanufacturing, and Pseudomonas is one of the organisms that does well in them. So this construct is a worked example of something my platform would surface as a need rather than a product: a cheap, specific detection and biocontrol reagent for contamination in residue-fed processes.

Sequence and provenance. The coding sequence is my own redesigned variant (LC-0.36), derived from the C-terminal receptor-binding region of T7 gp17. The parent sequence comes from the T7 tail fibre protein entry in UniProt.

Host. E. coli BL21(DE3). The parent protein is phage-derived and already native to an E. coli context, the DE3 lysogen supplies T7 RNA polymerase for the promoter I’ve chosen, and BL21 is protease-deficient (lon, ompT), which matters for a small domain that would otherwise be chewed up. It is also the cheapest and fastest route to the gram quantities a detection reagent would eventually need.

Localisation. Cytoplasmic and soluble, with an N-terminal His6 tag and a TEV protease site for purification. I don’t need secretion — this protein is harvested and purified, not exported — and keeping it cytoplasmic removes a whole layer of signal-peptide and translocation risk from a design that already has enough unknowns in it.

Part 2: Genetic parts

I haven’t hand-designed the backbone. pET-28a gives me replication, selection, lacI, and most of the expression furniture, which is a sensible thing to reuse.

One choice I’d defend rather than assume. The native pET RBS is strong, and strong is not automatically right — if the protein turns out to aggregate, dialling translation initiation down is one of the cleanest fixes available. I’ve checked the predicted initiation rate with the Salis RBS Calculator so that I know what I’d be tuning away from.

Part 3: Assembly

Order, 5’→3’, within the pET-28a backbone:

T7lac promoter → g10 RBS → ATG-His6 → TEV site → LC-0.36 RBD → stop → T7 terminator

Checks I ran on the assembled sequence:

• Reading frame intact from the start codon through to the stop.
• His6 and the TEV site in frame with the RBD, and the TEV site positioned so that cleavage leaves the intended N-terminus.
• No internal stop codons, particularly at the part junctions, which is where they tend to appear.
• Parts in the intended order and orientation on the rendered map.

Part 4: Optimisation and quality control

Codon optimisation. I optimised the RBD coding sequence for E. coli, applying constraints to avoid NdeI and XhoI sites (both in the pET-28a cloning region) and to keep GC content within a synthesisable window. Codon usage matters here because E. coli tRNA pools are strongly skewed: rare codons such as AGG, AGA and CUA can stall elongating ribosomes, and clusters of them are a common cause of truncated product and low yield. Worth noting that I deliberately did not aggressively optimise the first handful of codons — strong 5’ mRNA secondary structure suppresses initiation, and a slightly “worse” codon choice there often expresses better.

Sequence search. I ran my CDS against the nucleotide and protein databases to check that it is what I think it is, and to see how far my redesign has actually drifted from the natural parent.

Safety screen. The screen checks a construct against sequences of concern - homology to regulated pathogens and toxins, and to sequences flagged under harmonised biosecurity screening protocols — and reports anything that matches.

Part 5: From design to build

How I’d build it. The cassette is short — the RBD plus tag and linker is well under a kilobase, so I’d order it as a single synthetic DNA fragment and assemble it into linearised pET-28a by Gibson assembly. Gibson et al. (2009) showed that overlapping fragments can be joined isothermally in a single reaction, which removes the restriction-site planning that would otherwise constrain my part choices, and at this length there’s no reason to split it further.

What synthesis constrains. Chemical oligo synthesis carries a per-base error rate, and errors accumulate with length, so the practical limit isn’t what can be ordered but what can be ordered correctly. Carr et al. (2004) addressed exactly this with protein-mediated error correction, using mismatch recognition to enrich for correct products. My sequence is short enough that clone-and-sequence verification is cheaper than error correction, but if the design carried strong secondary structure or repeats I’d expect synthesis to struggle and would split it into shorter fragments.

Prototype versus scale. These genuinely diverge for me. Testing one RBD variant is a single fragment and one Gibson reaction. But the actual design question which of many RBD variants binds OprF best — is a library problem, and that’s where Kosuri et al. (2010) becomes the relevant route: oligos synthesised on high-fidelity microchips and selectively amplified from pools, which makes hundreds of variants affordable in a way that individually ordered fragments never would be. So the prototype is fragment-and-Gibson; the scale-up is chip-derived pools.

Part 6: What I don’t know yet

1. Whether the domain folds and binds outside the trimer. T7 tail fibres are homotrimeric, and the receptor-binding tip may well depend on that trimeric context to fold into a competent binding surface. Expressed as an isolated monomer, it could fold into something inert. How I’d find out: SEC-MALS to establish the oligomeric state of the purified protein, and binding measurement against purified OprF by BLI or ELISA. Design variant if it fails: fuse a T4 fibritin foldon domain to force trimerisation and re-test.

2. Whether it’s soluble at all in BL21. Phage structural proteins have a well-earned reputation for inclusion bodies, and a strong T7 promoter with a strong RBS is precisely the combination that drives aggregation. How I’d find out: small-scale expression trial, SDS-PAGE of soluble versus insoluble fractions. Variants to try: induction at 18°C, lower IPTG, a weaker RBS, or a SUMO or MBP solubility fusion.

3. Whether it’s specific enough to be useful. A detection reagent for a residue-fed fermentation has to distinguish P. aeruginosa from whatever else is growing in an unsterile agricultural feedstock. Binding OprF in a clean assay and binding it in that background are different claims. How I’d find out: a binding panel against related Pseudomonas species and against the mixed community from an actual residue-derived culture.

The honest summary is that the first of these would be the real bet. If the isolated domain doesn’t fold, everything downstream changes, and I’d know within one expression trial and one binding assay.

AI use

References

• Gibson, D. G. et al. Nature Methods 6, 343–345 (2009).
• Carr, P. A. et al. Nucleic Acids Research 32, e162 (2004).
• Kosuri, S. et al. Nature Biotechnology 28, 1295–1299 (2010).