Week 2 HW: DNA Design

Part 0: Set up in Kernel

The location of my files in Kernel is in the HTBAA26_Genspace repository, file name Paulina Liceaga

Part 1: Define your expression target

The target molecule is a single enzyme found in the Himalayan Mayapple plant, OMT3 or Pluviatolide O-methyltransferase, involved in the biosynthesis of etoposide, a chemotherapeutic compound of the topoisomerase inhibitor family (PubMed:26359402).

The accession code for this protein in UniProt is A0A0N9HMN6 and its genetic sequence is as follows:

MEMAPTMDLEIRNGNGYGDSGEELLAAQAHIYNHIFNFISSMALKCAVELNIPEILHNHQPKAVTLSELVQALQIPQAKSACLYRLLRILVHSGFFAITKIQSEGDEEGYLPTLSSKLLLKNHPMSMSPCLLGLVNPTMVAPMHFFSDWFKRSDDMTPFEATHGASLWKYFGETPHMAEIFNEAMGCETRLAMSVVLKECKGKLEGISSLVDVGGGTGNVGRAIAEAFPNVKCTVLDLPQVVGNLKGSNNLEFVSGDMFQFIPPADVVFLKWILHDWNDEECIKILKRCKEAIPSKEEGGKLIIIDMVVNDHNKGSYESTETQLFYDLTLMALLTGTERTETEWKKLFVAAGFTSYIISPVLGLKSIIEVFP

The enzyme would be expressed in an E.coli chassis and it’ll be produced in the cytoplasm. OMT3 is a soluble enzyme with no known membrane association or secretion signal — so no targeting sequence is required, which meaningfully simplifies this design compared to the P450 enzymes in the same pathway.

Part 2: Genetic parts

For the transcription start, I would use an inducible promoter, such as a T7- or lac-based promoter typical of a pET-style E. coli expression system. An inducible promoter lets the culture grow to a healthy density first, without the metabolic burden of constantly producing OMT3, and then lets expression be triggered on command at the time of the researcher’s choosing — giving more control than a constitutive promoter would.

For the translation start, I would use a ribosome binding site (RBS) of moderate-to-strong strength, a stronger RBS is a reasonable choice to maximize protein yield.

The coding sequence is the OMT3 gene itself: 1,119 base pairs (372 amino acids), including its start and stop codons, sourced from UniProt accession A0A0N9HMN6. This is the actual gene of interest, the sequence that gets transcribed and translated into the functional enzyme.

For targeting, no signal peptide or compartmentalization tag is needed, since OMT3 is a soluble, cytoplasmic enzyme with no known membrane association or secretion signal in its native plant context.

For the transcription stop, I would include a standard terminator (such as a T7 or rrnB terminator) immediately after the stop codon, to ensure a clean end to the transcript and prevent RNA polymerase from reading through into downstream vector sequence.

Finally, for the vector context, I would reuse an existing standard E. coli expression backbone (a pET-type vector, for example), which provides a ColE1-family origin of replication and a kanamycin resistance marker for selection. There’s no need to design a backbone from scratch here — an established, well-characterized backbone already provides everything this construct needs for replication and selection in E. coli.

Part 3 : Assembly

I originally added a T7 lac promoter with a BBa_B0034 Ribosome binding site and a T7 terminator but I then decided to substitute these individual parts for an already assembled backbone, so the construct consists of the pET-28a(+) backbone with the OMT3 CDS positioned for insertion at the vector’s MCS. After removing the redundant standalone promoter/RBS/terminator parts, the construct reduces to two elements: the backbone and the CDS. Kernel’s interface did not allow me to drag the CDS into the interior of the backbone’s MCS region, so in my current Kernel construct the CDS sits next to the backbone rather than literally inserted within it.

I confirmed the reading frame of the OMT3 CDS is intact (1,119 bp = 372 codons + stop, matching the UniProt-reported protein length exactly), with no accidental internal stop codons.

Part 4: PENDING

Part 5: Build

How would you build it? Direct synthetic gene synthesis of the codon-optimized OMT3 CDS as a gene fragment, followed by a standard one-step assembly (Gibson assembly, or restriction digestion/ligation using the BamHI/XhoI sites native to the pET-28a(+) MCS) into the vector. There’s no need for oligo-pool/chip-based synthesis, which is better suited to large combinatorial libraries rather than a single ~1.1 kb ORF.

What does synthesis constrain? A single moderate-GC, soluble-protein CDS like OMT3 is generally straightforward to synthesize, without the long hydrophobic repetitive stretches that make membrane-protein genes (like the pathway’s P450 enzymes) harder to synthesize. The main thing worth checking after codon optimization is whether any new restriction sites were accidentally introduced that would interfere with the BamHI/XhoI cloning strategy, or extreme local GC content that could complicate synthesis — neither is expected to be a major issue here.

Prototype vs. scale? Prototyping is this single synthesized CDS, cloned into the pET-28a(+) backbone and tested in a small BL21(DE3) culture. Producing DNA at meaningful scale (e.g., enough plasmid to seed a larger fermentation run) would not require re-synthesizing anything — it would instead mean standard plasmid maxi-prep amplification from a scaled-up overnight culture, since gene synthesis is a one-time design step rather than a per-batch production step.

Readings-

Part 6: What don’t you know yet?

I’d want to run an induction time-course (with and without IPTG) and quantify OMT3 protein levels (e.g., via SDS-PAGE or a His-tag-based Western blot) to confirm how much of the preotein is being expressed, and if it will express correctly to begin with.

Whether the N-terminal His-tag fusion affects OMT3 folding or activity. I assumed the tag would be a useful, low-impact purification handle, but some enzymes lose activity when fused to even small N-terminal tags. I’d want to express both the tagged and an untagged version and compare enzymatic activity (e.g., an in vitro assay measuring pluviatolide-to-bursehernin conversion) to confirm the tag isn’t interfering.

Whether the real location of the ori/KanR/lacI elements inside the pET-28a(+) block, as I’ve documented them from the standard reference map, actually matches Kernel’s specific copy of this part exactly. Since I couldn’t verify this directly within Kernel’s annotation tools, there’s a chance this specific library part has minor sequence differences from the commercial reference I cited. I’d want to BLAST the full Kernel sequence against the canonical pET-28a(+) reference to confirm an exact match before trusting my documented coordinates fully.