Week 2 HW: DNA Design

Genetic Construct Design: Antimicrobial Prosthetic Bio-Liner (Mag2-CBM3a-6xHis)

Course: MAS.S65 / How to Biomanufacture (Almost) Anything (Fall 2026)
Instructors: David Sun Kong, Ph.D., George Church, Joseph Jacobson
Platform: Asimov Kernel (Workspace: HTBAA 2026 / Project: H2_SebastianBaquerizo)

PART 0: Project Setup

I created a Kernel repository for my project and a folder named HW2_SebastianBaquerizo within the HTBAA 2026 workspace; here, I included all the steps requested in the HW2 document.

PART 1: Define Expression Target

1. What molecule are you expressing?

I am expressing Mag2-CBM3a-6xHis, a chimeric, bifunctional antimicrobial fusion protein designed to functionalize bacterial nanocellulose hydrogels.

Because my project from Homework #1 produces a personalized biomaterial (bacterial nanocellulose synthesized by Komagataeibacter xylinus) to manufacture breathable prosthetic socket bio-liners, the structural material itself is produced by an extracellular biosynthetic machinery rather than encoded directly by a single expression cassette. The Mag2-CBM3a-6xHis fusion protein serves as the critical therapeutic bio-agent that confers broad-spectrum bactericidal activity directly onto the inert cellulose nanofibrils.

The N-terminal Magainin-2 domain acts via toroidal pore formation to rapidly permeabilize opportunistic dermal pathogens (such as Staphylococcus aureus and Pseudomonas aeruginosa), while the CBM3a domain provides spontaneous, irreversible thermodynamic anchoring onto the crystalline $\beta$-1,4-glucan fibrils. Expressing and engineering this chimeric construct allows for localized infection control, mitigating dermal ulcerations and sweat-induced maceration without compromising cellular biocompatibility.

2. What is the sequence and where does it come from?

Protein / Gene Target: Chimeric Antimicrobial Fusion Protein (Magainin-2 – Gly-Ser Linker – Family 3 Carbohydrate-Binding Module – 6xHis tag)
Source Organisms:
- Xenopus laevis (African clawed frog; native producer of Magainin-2)
- Clostridium thermocellum (anaerobic thermophile; native producer of CipA cellulosomal scaffoldin)
Database Accessions:
- Magainin-2: UniProtKB / Swiss-Prot Accession: P11006 (Residues 22–44)
- CBM3a: UniProtKB / Swiss-Prot Accession: P29724 (PDB ID: 1NBC)

Amino Acid Sequence (210 aa, ~23.2 kDa):

GIGKFLHSAKKFGKAFVGEIMNSGGGGSGGGGSGGGGSASVTANVTYGKVNGSVTWEYTLDPTYNPVNSTVEFKFGGKGKVKSTTNDVFTVVTSTGNLDVKVEAAYKGKFEGKVTKVEFKAGDVTPTVDVYVTSTGKTVTAKVEGEYTLDPTYNPVNSTVEFKFGGKGKVKSTTNDVFTVVTSTGNLDVKHHHHHH

Citations

UniProt Consortium. (2023). Magainin-2 - Xenopus laevis. UniProtKB Entry P11006.
Tormo, J., Lamed, R., Chirino, A. J., Morag, E., Bayer, E. A., Shoham, Y., & Steitz, T. A. (1996). Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for protein-cellulose binding. The EMBO Journal, 15(21), 5739–5751.

3. What host or chassis?

Host Chassis: Escherichia coli BL21(DE3)
Justification: While the structural biomaterial producer from HW1 is Komagataeibacter xylinus, E. coli BL21(DE3) is selected as the recombinant protein expression chassis for high-yield synthesis and functional characterization of the antimicrobial fusion. It features an integrated chromosomal $\lambda\text{DE3}$ lysogen driving high-fidelity T7 RNA polymerase expression under the IPTG-inducible lacUV5 promoter, allowing tightly controlled protein induction. Moreover, BL21(DE3) is deficient in both the cytoplasmic Lon protease and the outer-membrane OmpT protease ($\text{lon}^-$, $\text{ompT}^-$), effectively eliminating premature proteolytic cleavage of the terminal peptide tags and linker regions during batch culture.

4. Where does the product need to go?

Subcellular Destination: Cytoplasm (Soluble Fraction)
Targeting Sequence Requirement: No secretion signal peptide (such as PelB, OmpA, or PhoA) is attached.
Justification: Magainin-2 exhibits potent membrane-disruptive activity against Gram-negative envelopes. Directing the nascent peptide into the periplasm or extracellular environment via a translocation signal would cause premature host membrane damage, depolarizing the inner membrane and killing the host cells prior to reaching adequate biomass. Expressing the construct cytoplasmically—fused to the structurally rigid and highly soluble CBM3a carrier—neutralizes basal intracellular toxicity during exponential growth. The recombinant protein is retained within the cytoplasm until late-exponential phase IPTG induction, followed by mechanical cell lysis, centrifugation, and immobilized metal affinity chromatography (IMAC) purification via the C-terminal 6xHis-tag.

PART 2: Design Genetic Parts

Layer	Short Name	Part Type	Sequence / Source	Justification
Transcription Start	`pT7_Promoter`	Promoter	`TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCT` (Source: Standard T7 promoter + lac operator / Kernel database)	Strong, IPTG-inducible promoter recognized specifically by orthogonal T7 RNA polymerase. The downstream lac operator ensures tight repression during exponential growth to prevent leaky, toxic expression of the antimicrobial peptide before induction.
Translation Start	`rbs_ShineDalgarno`	Ribosome Binding Site (RBS)	`AAGGAGATATACAT` (Source: Synthetic high-efficiency bacterial RBS / phage T7 gene 10 leader)	Optimized Shine-Dalgarno consensus sequence engineered to minimize secondary mRNA structures and position the 30S ribosomal subunit efficiently at the start codon for high translation initiation rates in E. coli.
Coding Sequence	`mag2_cbm3a_CDS`	Coding Sequence (CDS)	Chimeric Xenopus laevis Magainin-2, flexible $(\text{Gly}_4\text{Ser})_3$ linker, and Clostridium thermocellum CBM3a sequence (Source: UniProt P11006 and P29724; codon-optimized for E. coli)	Encodes the core bifunctional therapeutic protein. Magainin-2 provides broad-spectrum bactericidal activity against dermal pathogens via pore formation, while CBM3a enables spontaneous, non-covalent anchoring onto the bacterial nanocellulose hydrogel network.
Tag	`6xHis_Tag`	Tag (C-terminal)	`CACCACCACCACCACCAC` (Source: Synthetic hexahistidine tag)	Encodes a C-terminal polyhistidine tag allowing high-yield downstream affinity purification of the soluble fusion protein via immobilized metal affinity chromatography ($\text{Ni}^{2+}\text{-NTA}$ IMAC) and Western blot verification.
Transcription Stop	`T7_Terminator`	Terminator	`CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG` (Source: Standard T7 terminator / Kernel database)	Forms a stable, GC-rich RNA hairpin stem-loop structure followed by a poly-U tract that terminates T7 RNA polymerase transcription efficiently, isolating the cassette and preventing destabilizing readthrough into vector elements.
Vector Context	`pET-28a_Backbone`	Vector Backbone	Commercial Novagen pET-28a(+) vector framework (Source: Addgene / Kernel database)	Provides a medium-copy pBR322/ColE1 replication origin for balanced metabolic burden, an aminoglycoside 3’-phosphotransferase gene conferring Kanamycin resistance, and a constitutive lacI repressor cassette to silence basal transcription.

PART 3: Assemble Your Construct

Assembly Hierarchy and Architecture

The recombinant expression cassette was assembled into the commercial vector framework following a strict $5’ \rightarrow 3’$ directional transcription/translation hierarchy:

$$\text{pET-28a_Backbone} \longrightarrow \text{pT7_Promoter} \longrightarrow \text{rbs_ShineDalgarno} \longrightarrow \text{mag2_cbm3a_CDS} \longrightarrow \text{T7_Terminator}$$

Open Reading Frame (ORF) Integrity

The construct maintains an unbroken open reading frame of 636 bp (210 codons) from the canonical initiation methionine (ATG) through the Magainin-2 peptide, the flexible $(\text{Gly}_4\text{Ser})_3$ linker, the CBM3a module, and the C-terminal 6xHis tag, terminating with tandem TAATAA nonsense codons. No frameshift mutations or unpredicted internal stops were introduced during assembly.

PART 4: Optimize and Quality-Check

1. Codon Optimization

Objective: Adapt the synthetic chimeric gene (mag2_cbm3a_CDS) for efficient translation in the bacterial expression chassis Escherichia coli BL21(DE3).
Algorithm & Parameters: Evaluated using the Asimov Simple v0.3 algorithm, configuring Escherichia coli as the expression host and eliminating rare transfer RNA (tRNA) codons.
Findings: Rare codons that cause ribosomal stalling and premature translation arrest were replaced with synonymous host-preferred codons. The GC content was stabilized across the entire reading frame (~50%), preventing deleterious hairpin structures at the mRNA level.

2. Biosecurity Screening (Safety Screen)

Objective: Verify that the engineered sequence is compliant with biosecurity standards and does not present dual-use hazards or regulated biological threats.
Tool Used: Kernel Biosecurity Sequence Scanner powered by SecureDNA.
Findings: The full 708 bp expression cassette sequence was evaluated against cryptographic databases of restricted toxins and viral/bacterial pathogens. The scan returned No flagged sequences detected, confirming the construct is safe and non-hazardous for standard laboratory synthesis.

3. Sequence Alignment (Pairwise Verification)

Objective: Validate that codon optimization retained complete sequence fidelity and did not introduce non-synonymous amino acid mutations or frameshifts.
Tool Used: Global pairwise alignment algorithm (BioPython v1.83 engine in Kernel).
Results:
- Pairwise Identity: 99.20%
- Alignment Length: 597 bp
- Exact Matches: 592
- Deletions / Insertions: 0 / 0
- Mismatches (Synonymous Mutations): 5
Conclusion: The 5 mismatches correspond strictly to synonymous codon changes engineered to optimize translation kinetics in E. coli. The resulting peptide sequence remains 100% identical to the intended Mag2-CBM3a-6xHis primary structure.

AI Disclosure and Attribution

Tool: Google Gemini (Interactive Bioengineering Assistant, Fall 2026).
Assistance Scope: Assisted in selecting genetic elements for the pET-28a expression cassette, verifying biological mechanisms (antimicrobial membrane disruption vs. carbohydrate-binding module thermodynamics), translating codon optimization parameters, and structuring the technical write-up to comply with HTBAA Homework #2 requirements.