Peptide Production

This section outlines experimental design, strain and vector selection, part construction, cloning, protein purification, and in vitro/in vivo strategies.

1. Early Project Development: Strain and Vector Selection

1.1 Background: Why Phytochelatin-Based Systems?

Metal contamination poses severe risks to ecosystems and human health. While several physicochemical approaches exist for metal removal, they often lack specificity, generate secondary waste, or are economically unsustainable.

To address this, our project aimed to design a biological sequestration system using Escherichia coli engineered to synthesize phytochelatins (PCs) and metallothioneins (MTs)—two classes of natural metal-binding peptides.

Phytochelatins are synthesized enzymatically from glutathione (GSH) by phytochelatin synthase (PCS), forming γ-Glu–Cys polymers that chelate heavy metals through their thiol (-SH) groups. Complementary to this, metallothioneins are small, cysteine-rich proteins with high affinity for metals such as Cd²⁺, Pb²⁺, Hg²⁺, and Zn²⁺.

By combining these mechanisms, we aimed to engineer a versatile and modular detoxification system that could efficiently sequester multiple classes of heavy metals — from soft acids (e.g., Hg²⁺, Cd²⁺) to hard acids (e.g., Al³⁺, Fe³⁺).

1.2 Enzyme Mechanism and Pathway Overview

The phytochelatin biosynthetic pathway can be summarized as follows:

1. GSH synthesis:

   Glutathione (γ-Glu-Cys-Gly) is synthesized from amino acid precursors via two enzymatic steps:

   • γ-glutamylcysteine synthetase
   • glutathione synthetase

   To simplify circuit design, we utilized a bifunctional enzyme (GSHF) from Streptococcus thermophilus, which catalyzes both reactions within a single polypeptide.

2. Phytochelatin synthesis:

   PCS catalyzes the transfer of γ-Glu–Cys units from GSH to another GSH molecule, forming phytochelatin polymers (PC₂–PCₙ). The general reaction is:

   (γ-Glu–Cys)n​-Gly+GSH→(γ-Glu–Cys)n+1​-Gly+Gly

   PCS requires metal–GSH complexes as activators, linking its activity directly to metal exposure.

3. Metal sequestration:

   The resulting PCs and MTs bind metal ions through multiple cysteine thiol groups, forming stable complexes that can be either retained intracellularly or extracted post-expression for embedding into biofilters.

1.3 Strain Selection

We selected E. coli BL21(DE3) as the primary chassis due to its:

• Compatibility with T7 promoter-based expression systems.
• Minimal endogenous protease activity, improving recombinant protein stability.
• Extensive documentation and predictable behavior in high-copy expression systems.
• Proven success in expressing metal-binding and cysteine-rich proteins under controlled conditions.

Additionally, the BL21(DE3) strain carries a chromosomal copy of T7 RNA polymerase under lacUV5 control, which allows IPTG-inducible expression — a key feature for regulating potentially toxic metal-binding proteins.

1.4 Vector Selection and Expression Strategy

To ensure modular cloning and high-yield protein production, we used a pET-based expression system, optimized for T7-driven transcription.

This system allows tight IPTG control, minimizing background expression prior to induction and enabling stepwise optimization of metal exposure conditions.

Key considerations in vector design included:

• N-terminal 6×His tag for affinity purification via Ni–NTA chromatography.
• Thrombin cleavage site downstream of the tag to obtain tag-free protein for in vitro assays.
• Kanamycin selection marker to ensure plasmid stability during prolonged induction.
• RBS and spacer optimization for balanced co-expression in multi-gene constructs (e.g., PCS + GSHF).

For dual or multi-enzyme systems, separate expression cassettes were placed under independent T7 promoters and terminators to avoid transcriptional interference.

1.5 Metal-Binding Concept Validation

The early phase of our design involved computational analysis and literature validation to ensure feasibility:

• Protein structure data for PCS and MT were analyzed to confirm proper cysteine accessibility and folding under bacterial expression conditions.
• Homology modeling was used to identify key metal coordination sites and predict the impact of engineered cysteine-to-alanine substitutions in MT variants.
• Expression levels and codon usage were cross-checked for compatibility with E. coli codon bias.

These preliminary validations supported our hypothesis that engineered constructs could function synergistically within a bacterial chassis to chelate a broad range of metal ions.

2. BioBrick Design and Genetic Element Rationale

3. Choosing the Genetic Components and their Functional Description

The project includes the following modules and parts:

1. Phytochelatin Synthase Modules

Key Features:

• T7 promoter, lac operator, IPTG-inducible.
• 6xHis tag and thrombin cleavage site for purification.
• Spacers inserted to ensure proper folding and translation.
• Supports in vitro and in vivo characterization of PCS activity.

2. Glutathione Production Module

3. Metallothionein Modules

4. Metal Uptake Module

5. Regulatory and Spacer Elements

Design Rationale

• Modular design allows testing of individual components (PCS, MT, GSHF, MerP/T) and composite circuits.
• IPTG-inducible T7 promoters enable controlled expression to reduce cellular stress.
• His-tags and thrombin sites facilitate purification and functional assays.
• Spacers prevent steric hindrance and ensure proper folding and translation in multi-gene constructs.
• Combination of native and engineered MTs allows comparison of binding efficiency for soft vs. hard metals.

Characterization

• PCS and AtPCS: Soluble expression confirmed by SDS-PAGE and Western blot; phytochelatin synthesis measured using in vitro GSH supplementation.
• GSHF: Enzymatic production of glutathione confirmed via biochemical assays.
• Metallothioneins: Metal-binding capacity characterized using ICP-MS, AAS, or colorimetric assays.
• MerP/MerT: Assessed for intracellular Hg²⁺ uptake efficiency.
• Composite circuits: Evaluate intracellular phytochelatin production, metal sequestration, and tolerance in E. coli.

Applications

• Synthetic biology: Modular metal sequestration circuits.
• Environmental remediation: Heavy metal detoxification in bacterial chassis.
• Biochemical characterization: Study of enzyme kinetics and protein-metal interactions.
• Protein engineering: Comparison of native vs. rationally designed metallothioneins.

Stored gene fragments Wetlab Documentation

4. Genetic Circuit Construction and Design Strategy

Following the selection of individual components, we designed a series of modular circuits for testing metal remediation pathways.

5. In Vitro vs In Vivo Strategies

5.1 In Vitro Strategy

Goal: Produce and purify PCS enzymes (PsPCS or AtPCS) for controlled, cell-free phytochelatin synthesis.

Constructs used: Circuit 1 (PCS-only) and Circuit 5 (AtPCS-only)

Workflow: Express His-tagged PCS → incubate with GSH → optionally introduce metals to study PC-metal complex formation

Purpose: Quantitative and kinetic evaluation; production of PCs for embedding in biopolymer matrices

Future Work:

• Expression, purification, and tag removal of PCS
• Small-scale and bulk phytochelatin production assays
• RP-HPLC separation and DTNB quantitation

In-Vitro Phytochelatin Production Wetlab Documentation

5.2 In Vivo Strategy

Goal: Evaluate cellular metal detoxification in E. coli

Constructs used: Circuits 2, 3, 4, 6, 7

Workflow: IPTG induction → expose cultures to metals (Hg²⁺, Cd²⁺, Al³⁺) → assess PC or MT accumulation, metal removal, and tolerance

Purpose:

• Compare PCS- vs MT-mediated sequestration
• Study synergistic effects of MerP/T transporters
• Analyze impact of genetic configuration on detoxification

Future Work:

• Expression, purification, and metal-loading of MT variants
• In vivo quantification of intracellular PC/MT and metal-binding efficiency

5.3 Metallothionein Purification (native & engineered)

Future Work Protocol Highlights:

1. Expression in BL21(DE3)
2. Cell lysis and clarification
3. Ni²⁺–NTA affinity purification
4. His-tag removal (thrombin cleavage)
5. Apo-MT preparation with EDTA
6. Holo-MT preparation with stoichiometric metals
7. QC: Tricine SDS-PAGE, DTNB, ICP-MS/AAS
8. Storage at −80 °C

Gel Matrix Selection for Protein Immobilization

To immobilize and stabilize our proteins for water purification, we initially considered several hydrogel matrices, including PVA, chitosan, PEGDA, and sodium alginate. After evaluating their properties, we chose sodium alginate as our primary matrix due to its unique combination of biocompatibility, biodegradability, and mild gelation conditions.

Why alginate:

Sodium alginate is a natural polysaccharide extracted from brown seaweed, composed of mannuronic acid (M) and guluronic acid (G) residues. The M-to-G ratio controls gel texture — higher G content produces firmer gels, while higher M content yields softer gels. Alginate forms a 3D hydrogel network upon crosslinking with calcium ions (Ca²⁺), where the Ca²⁺ ions act as bridges between G-blocks, generating a porous, stable structure ideal for trapping contaminants.

Key properties:

• Biodegradable and environmentally safe
• Biocompatible and non-toxic
• Forms hydrogels under gentle conditions without harsh chemicals
• Porous network suitable for protein immobilization and contaminant adsorption

This combination of safety, tunable mechanical properties, and ease of gelation made alginate the optimal choice for our project.

Rationale for Bead Formation

Instead of casting bulk hydrogel sheets, we opted to form alginate beads. Bulk hydrogels tend to trap water within the polymer network, leading to swelling and reduced flow-through, which can hinder efficient contact with water contaminants. Beads, in contrast, provide high surface area, controlled porosity, and better mass transfer, allowing water to flow freely around and through the matrix. This format also facilitates easier handling, uniform protein immobilization, and modular deployment in filtration systems.

2. Sodium Alginate Bead Preparation & Storage Protocol Purpose:

Protocol 1 - using Sodium Alginate and CaCl2, without GDL

Materials & Reagents

• Sodium alginate powder (300 mesh/500 mesh)
• Calcium chloride dihydrate (CaCl₂·2H₂O)
• Phosphate-buffered saline (PBS) or deionized (DI) water
• Syringe (5–10 mL) with 18–22G needle or disposable dropper
• Beaker (100–250 mL) with magnetic stirrer + stir bar
• Sieve or sterile strainer
• Storage containers (tubes or bottles)
• Optional: Sodium azide (0.02% w/v) for long-term storage

Preparation of Solutions

1. Alginate solution (2% w/v)
   • Weigh 2 g sodium alginate.
   • Add to 100 mL PBS or DI water.
   • Stir on a magnetic stirrer until fully dissolved (1–2 h).
   • Gently warm (≤37 °C) if needed.
2. Calcium chloride solution (100–150 mM)
   • For 100 mM: dissolve 11.1 g CaCl₂·2H₂O in 1 L DI water.
   • Use 100–150 mM as a gelation bath.

Bead Formation Protocol

1. Pour 100–200 mL CaCl₂ solution into a beaker. Place on magnetic stirrer, stir gently (avoid turbulence).
2. Fill the syringe with alginate solution. Hold 5–10 cm above CaCl₂ bath.
3. Release droplets slowly into bath — beads form instantly. Repeat until desired amount is reached.
4. Curing: Leave beads in CaCl₂ bath for 15–30 min with gentle stirring.
5. Collection & Washing: Collect beads with sieve; wash 2–3× with PBS or DI water.
6. Storage: Transfer beads to container with PBS or DI water.
   • Store at 4 °C.
   • Replace buffer weekly.
   • For storage >1 month, add 0.02% sodium azide (do not use it for beads intended for protein encapsulation).
   • Do not freeze beads.

Notes & Tips

• Bead size control: Smaller needle gauge + higher drop height → smaller beads; larger gauge + lower height → larger beads.
• Prevent clumping: Gentle stirring during formation helps.
• Appearance: Beads should be spherical, translucent, and firm.
• Stability: Stable for weeks at 4 °C in PBS; with sodium azide, stable for months.

Gel Bead Preparation Wetlab Documentation

Purpose: Protocol 2 - using Sodium Alginate and CaCl2, with GDL

Protocol: Sodium Alginate Bead Preparation using GDL (Internal Gelation)

Aim: To prepare sodium alginate beads using internal gelation using CaCO₃ and glucono-δ-lactone (GDL) that slowly acidifies the medium, leading to a uniform increase of Ca²⁺ ions that can uniformly crosslink beads.

Why this method: This crosslinking strategy is centered around internal gelation and yields a more homogenous gel bead as compared to the rapid, external CaCl₂ protocol that focuses on quick crosslinking. This further provides increased structural stability and better filtration performance over time, which will enable longer filtration runs or attempts.

Materials and Reagents:

• Sodium alginate powder (medium viscosity)
• Calcium carbonate (CaCO₃) fine powder (food/pharma grade)
• Glucono-δ-lactone (GDL)
• Phosphate-buffered saline (PBS) or deionized (DI) water
• Syringe (5–10 mL) with 18–22G needle or disposable dropper
• Beakers (100–250 mL), magnetic stirrer
• Sieve
• Falcon tubes
• Optional (for >1 month storage of non-biological beads): Sodium azide (0.02% w/v)

Preparation of Solutions

1. Alginate Stock (2% w/v)

• Weigh 2 grams of sodium alginate and add to PBS or distilled water.
• Stir on a magnetic stirrer until fully dissolved (~1–2 hours).
• Gentle warming at 37 °C can be helpful.

2. Alginate + CaCO₃, working mix (prepare fresh every time before bead making)

• To 50 mL of 2% alginate, add 0.1–0.5% w/v CaCO₃ (i.e. 50–250 mg in 50 mL).
• Disperse thoroughly by slow stirring, avoiding the introduction of air or clumps.
• Start at 0.1% w/v CaCO₃ and gradually increase to 0.5% once the technique is mastered.

3. GDL Gelation Bath

• Dissolve 8.8–17.6 g GDL per liter of distilled water or PBS.
• Use 75–100 mM for faster, firmer gelation and 50 mM for slower, softer gels.
• Begin with 75 mM to obtain firm beads, then optimize up to 100 mM.
• Always prepare GDL freshly before every run to ensure consistency and avoid contamination.

Further Steps

1. Set up gelation bath

• Pour 100–200 mL of GDL solution into a beaker on a magnetic stirrer.
• Stir gently (need to finalize the RPM for this step); avoid turbulence to prevent bead fusion.

2. Load Alginate–CaCO₃ mix

• Fill a syringe or dropper with the freshly prepared mix.
• Hold the tip 5–10 cm above the GDL bath to ensure consistent entry onto the surface.

3. Prepare beads

• Release droplets slowly into the gently stirred GDL bath.
• Beads will begin crosslinking within minutes.
• Ensure beads do not touch each other to avoid fusion.

4. Curing Step

• Leave beads in the GDL bath for 30–60 minutes with gentle stirring.
• This time can be fine-tuned through multiple iterations.

5. Collection

• Collect beads using a sieve.
• Do not reuse the GDL bath for another batch — always use fresh GDL each time.

6. Washing / Neutralization

• Wash three times for 1 minute each with PBS or distilled water at near-neutral pH.
• Check pH before each wash step.
• Optional – For a firmer surface, rinse quickly (1–2 minutes) with 10 mM CaCl₂, then rinse again with PBS or distilled water.

7. Transfer and Storage

• Transfer beads into fresh PBS or distilled water (not the previous wash).
• Store at 4 °C.
• Replace the storage solution weekly and integrate peptides onto the gel beads within 2–3 days of preparation.
• For storage > 1 month without biological peptides, add 0.02% sodium azide to the storage solution (PBS or distilled water); note that this may reduce peptide integration efficiency.
• Do not freeze the beads.

Purpose: Protocol 2 - using Sodium Alginate and CaCl2, with GDL

Protocol: Sodium Alginate Bead Preparation using GDL (Internal Gelation)

Aim: To prepare sodium alginate beads using internal gelation with CaCO₃ and glucono-δ-lactone (GDL) that slowly acidifies the medium, leading to a uniform increase of Ca²⁺ ions that can uniformly crosslink beads.

Why this method: This crosslinking strategy is centered around internal gelation and yields a more homogenous gel bead compared to the rapid, external CaCl₂ protocol that focuses on quick crosslinking. This method provides increased structural stability and better filtration performance over time, enabling longer filtration runs or attempts.

Materials and Reagents:

• Sodium alginate powder (medium viscosity)
• Calcium carbonate (CaCO₃) fine powder (food/pharma grade)
• Glucono-δ-lactone (GDL)
• Phosphate-buffered saline (PBS) or deionized (DI) water
• Syringe (5–10 mL) with 18–22G needle or disposable dropper
• Beakers (100–250 mL), magnetic stirrer
• Sieve
• Falcon tubes
• Optional (for >1 month storage of non-biological beads): Sodium azide (0.02% w/v)

Additional Notes

1. For controlling the bead size
   • For smaller beads, use a smaller needle gauge or tip and release from a higher height above the surface of the gelation bath.
   • For larger beads, use a wider or flat-bottom needle tip and release from a lower height from the surface of the gelation bath.
2. Uniformity (key advantage of GDL): Internal gelation reduces the “skin-first” effect seen with CaCl₂ baths, resulting in more homogeneous cores.
3. Stoichiometry guidance: Aim for near-equimolar GDL:CaCO₃ to fully liberate Ca²⁺ (start with 1:1 molar; raise GDL if gels are too soft).
4. Bubble control: CaCO₃ can release CO₂ as pH drops. Minimize by using fine CaCO₃ and avoiding vigorous mixing.
5. Prevent clumping: Keep the GDL bath gently stirred during dripping and ensure there is no bead fusion by monitoring continuously.
6. Appearance & feel: Beads should be spherical, translucent, and resilient. If chalky or opaque, CaCO₃ concentration may be too high or under-dispersed.
7. Adjusting firmness in the entire bead:
   • Softer: lower CaCO₃ (0.1% w/v), GDL 50 mM, shorter cure (~30 min).
   • Firmer: higher CaCO₃ (0.3–0.5% w/v), GDL 75–100 mM, longer cure (~60 min).
8. Compatibility: For cell or protein encapsulation, avoid azide; tune GDL (≤50–75 mM) and rinse thoroughly to restore near-neutral pH.

3. Embedding the Peptides on the Beads

Background: Sodium alginate contains carboxyl groups (-COOH) on its mannuronic (M) and guluronic (G) acid residues. To covalently attach peptides, which have primary amine groups (-NH₂) (typically from lysine residues or N-terminal amines), we use EDC/NHS chemistry, a widely employed carbodiimide-based crosslinking method.

Step 1: Activation of Carboxyl Groups by EDC

• EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) is a water-soluble carbodiimide.
• In aqueous solution, EDC reacts with a carboxyl group on alginate to form a high-energy O-acylisourea intermediate:

Alginate - COOH + EDC → Alginate-COO-EDC (O-acylisourea intermediate)

Problem: The O-acylisourea is unstable in water and can hydrolyze back to the free carboxyl.

Step 2: Stabilization with NHS

• NHS (N-hydroxysuccinimide) is added to convert the unstable O-acylisourea into a stable NHS ester:

Alginate-COO-EDC + NHS → Alginate-COO-NHS + (EDC-urea byproduct)

The NHS ester is more resistant to hydrolysis and highly reactive toward primary amines.

Step 3: Peptide Coupling

• Peptides with free amine groups react with the NHS ester to form a covalent amide bond:

Alginate-COO-EDC + NHS + H₂N-Peptide → Alginate-CO-NH-Peptide + NHS

This reaction anchors the peptide onto the bead surface, with the carboxyl of alginate linked to the amino group of the peptide.

4. Rheological Analysis of Gel Beads (EXPECTED)

Rheological Literature Data for 2% Sodium Alginate Beads and 100 mM GDL

The rheological data for both 2% sodium alginate beads and 100 mM GDL (glucono-δ-lactone) systems that can be used for plotting shear stress vs. shear rate and storage/loss modulus relationships.

2% Sodium Alginate Rheological Data

Shear Stress vs Shear Rate Properties

Flow Behavior Characteristics:

• 2% sodium alginate solutions exhibit non-Newtonian shear-thinning behavior.
• Power-law model parameters for 2% alginate solutions:
• Consistency coefficient (k): 1.472 Pa·sⁿ
• Flow behavior index (n): 0.681 (< 1, confirming pseudoplastic behavior)
• R²: 0.977

Shear Rate Range and Response:

• Typical shear rate testing range: 0.1–500 s⁻¹.
• Exhibits shear-thinning behavior particularly in higher shear rate ranges (1–500 s⁻¹).
• Viscosity decreases significantly with increasing shear rate.

Storage and Loss Modulus Data

Dynamic Viscoelastic Properties for 2% Alginate:

• Storage modulus (G'): 5–50 Pa at 1 Hz frequency.
• Loss modulus (G''): 10–60 Pa at 1 Hz frequency.
• G'' > G' indicating viscous behavior predominates in solution state.
• Both moduli are frequency-dependent and increase with frequency.

Concentration Effects:

• 1% alginate: G' ≈ 5–15 Pa
• 2% alginate: G' ≈ 20–50 Pa
• Approximately 2-fold increase in storage modulus when concentration doubles.

Prepared Gel Beads Wetlab Documentation

Prepared Gel Beads Wetlab Documentation

Alginate Hydrogel Beads Mechanical Properties

Elastic Moduli Ranges:

• Calcium alginate hydrogels: 1-100 kPa elastic modulus range.
• 2% alginate with CaCl₂: 5-20 kPa depending on crosslinking conditions.
• Young's modulus for alginate beads: 250-900 kPa depending on formulation.

Shear rate v/s Shear stress (in beads without GDL) Wetlab Documentation

Storage/Loss Modulus for Gel Beads:

• G' values: 100-1000 Pa for crosslinked alginate systems.
• G'' values: 10-200 Pa for crosslinked systems.
• G' > G'' in gel state, indicating elastic behavior dominates.

Storage / Loss modulus for gel beads Wetlab Documentation

100 mM GDL Rheological Data

Gel Formation Characteristics:

• GDL concentration: 100 mM (typical range 50-150 mM).
• pH reduction: From ~6.5 to ~4.5–5.0 during hydrolysis.
• Gelation kinetics: Time-dependent gel formation over 30–120 minutes.

Shear Stress vs Shear Rate for GDL Systems:

• Pre-gelation: Newtonian or slightly shear-thinning behavior
• During gelation: Increasing yield stress development
• Post-gelation: Non-linear viscoelastic behavior with yield stress.

Shear rate v/s Shear stress (in beads with GDL) Wetlab Documentation

Storage and Loss Modulus

Dynamic Moduli During GDL Gelation:

• Initial G' (pre-gel): 0.1–1 Pa
• Gel point G': 1–10 Pa (when G' = G'')
• Final G' (mature gel): 10–100 Pa
• Final G'': 1–20 Pa
• Gelation half-time: 32–45 minutes at room temperature.

Storage / Loss modulus for GDL gelation Wetlab Documentation

5. Result

• Alginate beads now present peptides covalently bound on their surfaces.
• The peptides’ metal-binding residues remain accessible, allowing the beads to function as micro-reactors, capturing toxic ions such as Cd²⁺, Pb²⁺, or Hg²⁺ from water.