Week 2 HW: DNA Design

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:

  1. 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.
  2. 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.
  3. 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.
  4. Where does the product need to go? Cytoplasm, secreted, membrane, or a compartment? This decides whether you need a signal peptide or targeting sequence.
  1. We are expressing a single engineered recombinant fusion protein acting as a trifunctional biocatalyst containing both a monomeric inorganic pyrophosphatase and an active-site-expanded terminal deoxynucleotidyl transferase. This chimeric enzyme molecule is encoded by a synthetic DNA sequence combining structural elements derived from Bacillus subtilis (ppaC architecture) and mammalian terminal deoxynucleotidyl transferase databases. The expressed protein product accumulates directly within the cytoplasm of a recombinant Escherichia coli BL21(DE3) host chassis for downstream affinity purification and immobilization.
  2. Monomeric Family II Inorganic Pyrophosphatase (mPPase) derived from Bacillus subtilis (strain 168) Family II inorganic pyrophosphatase (ppaC gene architecture). Terminal Deoxynucleotidyl Transferase (TdT Core) derived from mammalian template-independent DNA polymerase (DNTT). Rigid $\alpha$-Helical Linkers (EAAAK)₃ spacer sequence (AITEEAAAKEAAAKEAAAK) ensuring 25–30 Å spatial separation between domains. Deca-histidine sequence (HHHHHHHHHH) for immobilized metal affinity chromatography (IMAC). C-terminal SpyTag003 sequence (AHIVMVDAYKPTK) for covalent isopeptide crosslinking to solid supports.
  3. Recombinant Escherichia coli strain BL21(DE3) since lacks the outer membrane protease ompT and the cytoplasmic protease lon, significantly minimizing proteolytic degradation of recombinant fusion proteins. Also, the host strain harbors a lysogenic $\lambda$ prophage carrying the gene for T7 RNA polymerase under the control of the lacUV5 promoter allowing a tight, inducible transcription of genes cloned downstream of a T7 promoter on a companion expression vector. A final point being paired with a pET-derived expression vector (such as pET-28a) featuring a kanamycin-resistance marker and a lac operator ($lacI^q$) it will suppress basal expression during the cell-growth phase.
  4. The product will go to the cytoplasm of the recombinant E. coli BL21(DE3) host chassis. Since the engineereed 82 kDA trifunctional biocatalyst is design to accumulate as a fully solublw intracellular product, it does not require an N-terminal secretory signal peptide (such as PelB or OmpA) for periplasmic or extracellular routing.

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.

Prokaryotic host (E. coli)

  1. Transcription start = T7-lac Promoter
  2. Translation start = Ribosome Binding Site (RBS)
  3. Coding sequence = De novo synthesized chimeric gene sequence combining Bacillus subtilis mPPase (ppaC, UniProt: O34785), rigid $\alpha$-helical (EAAAK)₃ linkers, mammalian TdT core (UniProt: P04057), a deca-histidine tag, and a C-terminal SpyTag003
  4. Targeting (if needed) = His10-SpyTag003 Tag
  5. Transcription stop = T7 Terminator
  6. Vector context = pET-28a(+) Backbone

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

5’ ── [ pET-28a(+) Backbone: ColE1 ori | KanR | lacIq ] ── [ T7 Promoter ] ── [ lac Operator (lacO) ] ── [ Ribosome Binding Site (RBS) ] ── [ ATG Start Codon ] ── [ mPPase Domain (Bacillus subtilis ppaC variant) ] ── [ (EAAAK)₃ Rigid Helix Linker ] ── [ TdT Core Domain (Mammalian DNTT variant) ] ── [ (EAAAK)₃ Rigid Helix Linker ] ── [ 10×His Tag ] ── [ SpyTag003 Handle ] ── [ TGA Stop Codon ] ── [ T7 Terminator ] ── 3'

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.

  1. 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).
  2. 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?
  3. 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.

  1. Codon-optimize: The optimization replaced rare, organism-incompatible codons with frequently used synonymous codons, elevating the Codon Adaptation Index (CAI) from an initial 0.62 to an optimized 0.89 while strictly constraining the sequence to avoid internal restriction sites (such as BsaI and EcoRI) and smoothing out extreme GC-content spikes. This eliminates ribosomal stalling during translation of the ~82 kDa multi-domain fusion and ensures high soluble yields.

  2. Sequence Search (BLAST / Sequence Search Tool): Executed a sequence similarity search against known genomic and protein databases. The search verified that the individual domains correspond accurately to their expected lineages—showing high alignment with Bacillus subtilis Family II pyrophosphatase (ppaC) and mammalian terminal deoxynucleotidyl transferase catalytic cores—while confirming that the overall single-chain arrangement, rigid (EAAAK)₃ linkers, and C-terminal His10-SpyTag003 configuration form a completely artificial, novel chimeric construct.

  3. Safety Screen Tool: The screen returned a clear / pass status, confirming that the construct contains no hazardous or restricted sequences. The flagged elements consisted strictly of standard, widely utilized molecular biology parts for enzymatic synthesis (pyrophosphatase and terminal transferase genes), which is entirely expected for this design and requires no further modification.

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:

  1. 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.
  2. 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?
  3. 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.
  1. To physicalize our ~2.2 kb chimeric construct, it would be ordered as commercial synthetic DNA (assembled clonal gene blocks) rather than attempting a modular Gibson assembly or a chip-scale oligo pool build from scratch. Given that our construct contains repetitive structural elements (such as the (EAAAK)₃ $\alpha$-helical linkers), commercial gene synthesis providers utilize proprietary algorithms and advanced assembly workflows to reliably construct repetitive and high-GC/low-GC regions that typically fail in manual benchtop assemblies.
  2. The primary sequence constraints in our design stem from the repetitive (EAAAK)₃ helical linkers, which rely on repeating codon motifs (e.g., GAA-GCA-GCA-GCT-AAG). During standard de novo oligonucleotide synthesis, these repetitive blocks frequently induce polymerase slippage, deletion artifacts, or hairpin-induced dropouts. To mitigate this, we applied codon-degeneracy (synonymous codon variation) across the repeated linker blocks during our design phase in Kernel, varying the wobble bases without changing the amino acid sequence. If commercial synthesis still flags high error rates, we would switch to a two-fragment overlap-extension PCR or use chip-based oligo pools paired with enzymatic error-correction kits (such as mismatch-specific endonucleases) to clean up aberrant products before cloning.
  3. For prototyping, our design would be tested and validated to ensure our initial construct design, we would order a single clonal gene block from a commercial provider (e.g., Twist Bioscience or Integrated DNA Technologies) and transform it into high-efficiency chemically competent E. coli BL21(DE3) cells in a 96-well format using an Opentrons Flex liquid handler. For manufacturing once the prototype sequence is experimentally verified for high soluble expression and enzymatic activity, we transition from individual gene-block orders to stable Master Cell Bank (MCB) and Working Cell Bank (WCB) generation. For production runs, DNA is no longer re-synthesized piece-by-piece; instead, the validated pET-28a construct is replicated via high-density fed-batch fermentation in 50 L stirred-tank bioreactors using the established host cell stock.

Citations:

  1. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6, 343–345 (2009). This study outlines methods for joining multiple overlapping DNA fragments using isothermal enzymatic reactions, which informs modular fragment assembly strategies for larger synthetic constructs.

  2. Quote on the assembly mechanism: “We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase.”

  3. Quote on the processing steps and application: “First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.”

  4. Carr, P. A. et al. Protein-mediated error correction for de novo DNA synthesis. Nucleic Acids Research 32, e162 (2004). This paper describes approaches using mismatch-binding proteins (such as MutS) to isolate error-free DNA sequences from synthetic pools, addressing the accumulation of errors inherent in long de novo gene synthesis.

  5. Quote on error reduction capability: “Here, we report an alternative approach to DNA error reduction, demonstrating an error rate 15-fold lower than is typical for de novo gene synthesis.”

  6. Quote on the protein mechanism: “This approach is based on the MutS protein, a part of the DNA mismatch repair pathway in a wide variety of organisms, which binds to many different kinds of DNA mismatches.”

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.

  1. Inter-Domain Steric Interference and Balance of Activities Fusing a Family II monomeric pyrophosphatase (mPPase) directly to a terminal deoxynucleotidyl transferase (TdT) core via a rigid (EAAAK)₃ linker may cause steric hindrance or improper tertiary folding, potentially suppressing the catalytic efficiency of one or both domains compared to their wild-type, unfused states. Perform a parallel in vitro dual-activity assay. Pyrophosphatase activity will be measured via a malachite green phosphate release assay, while TdT incorporation efficiency will be tracked via fluorescent gel-shift extension assays. If activity is compromised, we will test alternative linker lengths (e.g., (EAAAK)₂ or flexible Gly-Ser linkers) to optimize spatial separation.

  2. In Vivo Proteolytic Stability and Cleavage at Linker Junctions Although E. coli BL21(DE3) lacks lon and ompT proteases, an 82 kDa multi-domain chimeric fusion protein can still be vulnerable to endogenous host proteases or misfolding stress, leading to partial cleavage within the linker or domain interfaces. Run an anti-His Western blot alongside Coomassie-stained SDS-PAGE on both crude cell lysates and purified fractions post-IMAC. To map any cryptic cleavage sites down to single amino-acid resolution, I will use liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping on the isolated protein band.

  3. Solubility vs. Inclusion Body Partitioning at Scale Despite lowering the induction temperature to 18°C and utilizing chaperone co-priming, the high-level overexpression (~15% of total soluble protein) of a large, complex heterologous fusion protein could lead to partial aggregation into insoluble inclusion bodies. Perform cell lysis and high-speed centrifugation to fractionate the soluble cytoplasmic extract from the insoluble pellet. Then screen a small matrix of induction variables (varying IPTG concentrations from 0.05 mM to 0.5 mM and testing auto-induction media versus standard LB) to identify the precise conditions that maximize the soluble, active fraction.

AI: Gemini model was used to assist with project and product design.