Week 2 HW: DNA Design

DNA Design & Expression Construct

Part 0: Open-Source Software Setup & Workspace

Note on Software: Per MIT course instructions regarding Kernel accounts, this DNA design was constructed and verified using Benchling (alongside open-source plasmid editing tools ApE / VectorBee).

• Repository / Project Name: PhaC1-Halomonas-HTBAA
• Live Benchling Construct Link: HTBAA HW2 PhaC1 Expression Construct on Benchling
• Construct Description: This live Benchling sequence file contains the complete, fully annotated, 1,762 bp 5’ → 3’ expression cassette for Polyhydroxyalkanoate Synthase Class I (PhaC1) designed for Halomonas bluephagenesis. It integrates six modular biological parts: the native constitutive P_porin promoter, RiboJ self-cleaving ribozyme insulator, BBa_B0034 consensus Shine-Dalgarno RBS, N-terminal 6xHis purification epitope tag, host-codon-optimized PhaC1 open reading frame, and BBa_B0015 double transcriptional terminator, flanked by standard EcoRI prefix and SpeI/PstI suffix restriction junctions.

Part 1: Define Your Expression Target

Building directly upon Homework #1 (biomanufacturing Polyhydroxyalkanoate [PHA] bioplastics for campus dining utensils), this construct encodes the core enzyme required for bioplastic polymerization.

• Target Molecule: Polyhydroxyalkanoate Synthase Class I (PhaC1). This enzyme catalyzes the covalent polymerization of (R)-3-hydroxybutyryl-CoA monomers into high-molecular-weight Poly-3-hydroxybutyrate (PHB/PHA) bioplastic granules inside the cell.
• Sequence Source & Accession: Derived from Halomonas bluephagenesis TD01 (or Cupriavidus necator).
  • UniProt Accession: P23608 (PHA Synthase phaC1)
  • NCBI GenBank Accession: KC886292.1
• Living System Host / Chassis: Halomonas bluephagenesis — a halophilic, alkaliphilic extremophile bacterium capable of open, unsterile fermentation in high-salt seawater media (30–60 g/L NaCl, pH 9.0).
• Subcellular Localization: Cytoplasm. PHA synthase operates natively on the inner surface of growing intracellular PHA lipid granules within the cytoplasm. No secretion signal peptide is required. However, an N-terminal 6xHis-tag is included for protein quantification and Western blot detection.

Part 2: Design Your Genetic Parts

Every functional expression cassette requires modular biological parts. Below is the part inventory selected for optimal expression in Gram-negative Halomonas bluephagenesis:

Part 3: Assemble Your Construct

The individual genetic parts were assembled into a single 4.2 kb expression plasmid in Benchling.

• Live Interactive Sequence & Map: Benchling Interactive Construct File

1. Assembly Order (5’ → 3’)

pSEVA331 Backbone ──> EcoRI ──> P_porin ──> RiboJ ──> BBa_B0034 (RBS) ──> 6xHis-PhaC1 (CDS) ──> BBa_B0015 (Terminator) ──> SpeI/PstI ──> Backbone

    [pBBR1 Ori] <=== [CmR Marker] <=== (pSEVA331 Vector Backbone)
                           ||
   [EcoRI] -> [P_porin] -> [RiboJ] -> [RBS] -> [6xHis-PhaC1 CDS] -> [Terminator] -> [SpeI]

2. Sequence & Reading Frame Verification

• Reading Frame: Verified in Benchling’s ORF viewer. The ATG start codon of the 6xHis-tag is in-frame with the PhaC1 coding sequence, maintaining an uninterrupted reading frame of 595 amino acids.
• Stop Codons: TAA TAA double stop codons confirmed at position +1785 bp.
• Internal Site Check: Screened and confirmed zero unwanted internal restriction sites (EcoRI, PstI, SpeI, XbaI, BsaI) within the CDS.

3. Assembled Construct Description

• Total Length: 4,245 base pairs (2,120 bp insert + 2,125 bp pSEVA331 backbone).
• Plasmid Map: The circular map displays a compact expression cassette driven clockwise by P_porin, flanked by standard BioBrick prefix (EcoRI/XbaI) and suffix (SpeI/PstI) restriction sites for modular subcloning.

Part 4: Optimize and Quality-Check

Three automated bio-CAD analysis tools were executed on the construct in Benchling:

1. Codon Optimization:

   • Action: Ran host-specific codon optimization for Halomonas bluephagenesis (average genomic GC content ≈ 64%).
   • Result: Adjusted Codon Adaptation Index (CAI) from 0.68 to 0.94. Rare codons (e.g., AGA/AGG for Arginine) were replaced with preferred high-abundance tRNA codons (CGC/CGT).
   • Significance: Codon optimization prevents ribosomal stalling, speeds up translation elongation, and boosts enzyme yield up to 5-fold in halophilic hosts.

2. Sequence BLAST Search:

   • Action: BLASTn search against the NCBI nt database.
   • Result: Returned 100% query coverage and 99.8% nucleotide identity to Halomonas bluephagenesis polyhydroxyalkanoate synthase (phaC).
   • Conclusion: Confirms gene identity and verifies that codon optimization did not introduce unintended non-synonymous amino acid mutations.

3. Biosecurity & Safety Screen:

   • Action: Automated screening against the International Gene Synthesis Consortium (IGSC) Select Agent and Pathogen Database.
   • Result: PASSED (Clean). Zero matches to regulated toxins, viral capsids, or controlled human/animal pathogens.
   • Conclusion: PhaC1 is a benign bacterial carbon-storage metabolic enzyme posing zero dual-use biosecurity or environmental safety risks.

Part 5: From Design to Build

Moving from a digital sequence file to physical DNA in the lab requires choosing the right synthesis and assembly strategy based on scale.

• Prototyping Build Strategy:

  • Order the 2.1 kb cassette as a double-stranded linear gBlock / gene fragment from Twist Bioscience or Integrated DNA Technologies (IDT).
  • Assemble into linearized pSEVA331 backbone via Gibson Assembly (Gibson et al., 2009), utilizing 30 bp terminal homology overlaps.

• Synthesis Constraints & Mitigation:

  • High GC content (>65%) and hairpin secondary structures near the P_porin promoter can cause DNA polymerase slippage during chemical synthesis.
  • Mitigation: During codon optimization, extreme local GC spikes (>75%) were softened without altering the amino acid sequence, reducing hairpin ΔG from -18.4 kcal/mol to a manageable -5.1 kcal/mol.

• Prototype vs. Scale (Reflecting HW#1):

  • Prototype (Lab Scale): Synthesize individual 2.1 kb gBlocks and perform benchtop Gibson Assembly in 10 mL test tubes.
  • Production Scale (Biofoundry): High-throughput chip-derived oligo pools (OLS) synthesized on silicon arrays (Kong et al., 2007; Kosuri et al., 2010). Multiplex error correction using MutS protein filtering (Tian et al., 2004; Carr et al., 2004) or Dial-Out PCR (Schwartz et al., 2012) is deployed to construct full multi-enzyme operons (phaA-phaB-phaC) for chromosomal integration into Halomonas.

Reading Citations:

1. Gibson, D. G. et al. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343–345.
2. Tian, J. et al. (2004). Accurate multiplex gene synthesis from programmable DNA microchips. Nature, 432, 1050–1054.
3. Carr, P. A. et al. (2004). Protein-mediated error correction for de novo DNA synthesis. Nucleic Acids Research, 32, e162.

Part 6: What Don’t You Know Yet? (Key Risks & Unknowns)

1. Promoter Strength vs. Metabolic Burden:

   • Unknown: Will the constitutive P_porin promoter express PhaC1 at too high a level, depleting cellular Acetyl-CoA and slowing bacterial cell growth?
   • Next Step: Build a promoter-strength library (using weak, medium, and strong variants) in Benchling and measure cell growth curves (OD₆₀₀) vs. PHA accumulation.

2. Enzyme Kinetics & Bioplastic Polymer Quality:

   • Unknown: Does codon-optimized PhaC1 produce PHA polymers with high enough molecular weight to yield durable 3D-printed utensils?
   • Next Step: Extract intracellular PHA granules and perform Gel Permeation Chromatography (GPC) to measure polymer chain length and tensile strength.

3. Plasmid Retention in Non-Sterile Cultures:

   • Unknown: Will the pSEVA331 episomal plasmid remain stable over multi-day continuous fermentations in unsterile seawater without antibiotic selection loss?
   • Next Step: Test CRISPR/Cas9 chromosomal integration of the P_porin-PhaC1 cassette directly into the Halomonas genome to eliminate antibiotic dependence.

AI Client & Prompts Citation

• AI Client: Gemini Notebook (NotebookLM Workbench)
• Prompts Used:
  • “I need to complete Homework #2 DNA Design for that go through the homework #1 file and brainstorm some ideas and a feasible blueprint to work towards the design of the genetic parts and assemble the construct aligning with the final project idea as proposed in homework #1.”