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Wetlab Experimentation

The wet lab work for Project POSEIDON focused on the molecular cloning and verification of genetic constructs designed for metal-binding peptide expression. Using standardized synthetic biology workflows, we employed the pET28b(+) expression system and E. coli BL21(DE3) chassis to assemble and confirm our engineered sequences. The following section outlines the experimental protocols followed during this process.

Protocol: Preparation of Inoue Transformation Buffer

Objective:
To prepare a high-efficiency transformation buffer (Inoue et al. method) for the generation of chemically competent E. coli cells.

Reagents and Composition (for 50 mL final volume):

ReagentAmountPurpose
MnCl₂·4H₂O0.544 gEnhances membrane permeability
CaCl₂·2H₂O0.110 gStabilizes cell membrane
KCl0.9325 gMaintains ionic strength
PIPES buffer (0.5 M, pH 6.7)1.0 mLProvides buffering capacity
KOH (0.28 g/mL stock)Added dropwiseAdjusts pH to 6.7
Deionized waterTo 50 mLSolvent

Procedure:

  1. Add approximately 30 mL of deionized water to a clean 100 mL beaker or flask.
  2. Dissolve the salts sequentially under continuous stirring:
    • MnCl₂·4H₂O
    • CaCl₂·2H₂O
    • KCl
  3. Add 1.0 mL of 0.5 M PIPES buffer (pH 6.7) and mix thoroughly.
  4. Adjust the pH to 6.7 by adding KOH (0.28 g/mL stock) dropwise while monitoring with a calibrated pH meter.
  5. Bring the final volume up to 50 mL with deionized water.
  6. Filter sterilize the solution using a 0.22 µm membrane filter into a sterile container.
  7. Store the buffer at 4 °C until use.

Rationale:

The presence of Mn²⁺ and Ca²⁺ ions enhances the permeability of the E. coli cell membrane, facilitating DNA uptake during transformation. Maintaining low temperatures helps preserve membrane integrity and improve transformation efficiency.

Protocol: Preparation of Chemically Competent E. coli Cells (Inoue Method)

Objective:
To prepare high-efficiency chemically competent E. coli cells using the Inoue transformation buffer for DNA uptake and transformation experiments.

Materials and Reagents:

Procedure:

A. Overnight Culture Setup

  1. Prepare two 25 mL LB broth tubes:
    • Tube A (5 mL): Sterility control (no inoculation).
    • Tube B (20 mL): Inoculate with a single E. coli colony from a fresh plate.
  2. Incubate both tubes overnight at 37 °C with shaking at 180 rpm.

B. Log-Phase Growth

  1. Dilute the overnight culture into 100 mL fresh LB broth in a 250 mL flask.
  2. Incubate at 37 °C, 180 rpm.
  3. Monitor growth by measuring OD₆₀₀ periodically.
  4. When the OD₆₀₀ reaches 0.3–0.4 (mid-log phase), place the culture flask immediately on ice.

C. Harvesting and Washing

  1. Divide the chilled culture into two 50 mL Falcon tubes.
  2. Centrifuge at 2500 × g for 10 min at 4 °C using a flat rotor (preferred for soft, even pellets).
  3. Carefully discard the supernatant without disturbing the pellet.
  4. Gently resuspend each pellet in 5 mL ice-cold Inoue Transformation Buffer (ITB).
  5. Combine both suspensions into a single Falcon tube (10 mL total) and add additional ITB to a final volume of 35 mL.
  6. Centrifuge again at 2500 × g for 10 min at 4 °C.
  7. Remove the supernatant completely.
  8. Resuspend the final pellet in 1.5 mL ITB.
  9. Add 112.5 µL DMSO slowly with gentle mixing to avoid cell lysis.
  10. Prepare 50 µL aliquots in sterile MCTs (approximately 15 tubes).
  11. Snap-freeze the aliquots in liquid nitrogen and store at –80 °C for long-term use.

Rationale:
Competent cell preparation under cold conditions minimizes membrane fluidity and damage, allowing metal ions from ITB (Mn²⁺, Ca²⁺, K⁺) to integrate efficiently into the membrane. This enhances DNA uptake during transformation. DMSO acts as a cryoprotectant, preventing cell damage during freezing and storage.

Protocol: Transformation of competent E. coli cells with pET28b(+) vector to check for competency, and make glycerol stocks

Objective:
To introduce the pET28b(+) expression plasmid into competent E. coli cells via heat-shock transformation for subsequent selection and expression studies.

Materials and Reagents:

Procedure:

  1. Plasmid Preparation:
    • The pET28b(+) plasmid stock (206 ng/µL) was diluted with 2 µL nuclease-free water to obtain a working concentration of ~103 ng/µL.
    • For transformation, 25 ng plasmid DNA (0.24 µL of the diluted solution) was used.
  2. Transformation:
    • Thaw competent E. coli cells on ice.
    • Add 0.24 µL of the plasmid DNA to 50 µL of competent cells.
    • Mix gently by flicking; do not pipette up and down.
    • Incubate the mixture on ice for 30 minutes.
    • Perform heat shock at 42 °C for 45 seconds, then immediately place on ice for 2 minutes.
    • Add 900 µL of pre-warmed LB broth to the tube.
    • Incubate for 1 hour at 37 °C, shaking at 180 rpm to allow recovery and antibiotic resistance expression.
  3. Plating and Selection:
    • Plate 100–200 µL of the recovery culture on LB + Kanamycin (50 µg/mL) agar plate.
    • Incubate overnight at 37 °C.
    • Observe colony formation.

Rationale:

Heat-shock transformation allows plasmid DNA uptake through transient pores formed in the cell membrane. The recovery step enables expression of the Kanamycin resistance gene before selection on antibiotic plates.

Objective:
To prepare stable glycerol stocks of E. coli transformants harboring the pET28b(+) plasmid for long-term storage at –80 °C.

Materials and Reagents:

Procedure:

  1. Pick a well-isolated single colony from the LB + Kanamycin plate using a sterile inoculating loop.
  2. Inoculate into 6 mL LB broth containing 6 µL Kanamycin.
  3. Incubate at 37 °C, shaking at 180 rpm for 12–14 hours to obtain an overnight culture.
  4. Mix 500 µL of the overnight culture with 500 µL of sterile 50% glycerol in a cryovial.
  5. Vortex gently or pipette up and down to ensure uniform mixing.
  6. Label the vial with strain name and plasmid details.
  7. Store the prepared glycerol stock at –80 °C for long-term preservation.

Rationale:

Glycerol acts as a cryoprotectant, preventing ice crystal formation and maintaining cell viability during freezing. Glycerol stocks allow long-term preservation of genetically stable clones for future plasmid isolation or protein expression studies.

Protocol: Plasmid Isolation and Quantification

To culture E. coli transformants harboring the pET28b(+) plasmid, isolate plasmid DNA using the alkaline-lysis method followed by spin-column purification, and quantify the purified DNA using a NanoDrop spectrophotometer.

A. Culture Preparation

Materials:

Procedure:

  1. Inoculate 20 mL LB broth with 40 µL glycerol stock and 20 µL Kanamycin in a sterile 100 mL flask.
  2. Incubate at 37 °C, 180 rpm for approximately 14 hours until turbid growth is observed.

B. Culture Harvest

  1. Transfer the overnight culture to a 50 mL Falcon tube.
  2. Centrifuge at 4500 rpm for 3 min at room temperature to pellet the cells.
  3. Carefully decant the supernatant without disturbing the pellet.

C. Resuspension and Lysis

(Keep samples on ice unless otherwise specified.)

  1. Add 250 µL ice-cold P1 buffer to the pellet. Resuspend thoroughly by pipetting and gentle vortexing until homogeneous.
    Rationale: P1 buffer resuspends cells and contains RNase A to reduce RNA contamination. Cold handling prevents premature lysis.
  2. Transfer the suspension to a 1.5 mL microcentrifuge tube.
  3. Add 250 µL P2 buffer and mix gently by slow inversion for 4–6 minutes. Avoid vigorous vortexing.
    Rationale: P2 (NaOH + SDS) lyses cells and denatures DNA and proteins. Gentle inversion ensures uniform lysis while minimizing genomic DNA shearing.
  4. Add 350 µL N3 buffer and invert immediately several times until the solution becomes colorless and a white precipitate forms.
    Rationale: N3 neutralizes the alkaline mixture and precipitates cellular debris and SDS complexes; plasmid DNA remains in the cleared supernatant.

D. Clarification and Binding

  1. Centrifuge at 13 000 rpm for 10 min at room temperature to pellet debris.
  2. Transfer the clear supernatant to a spin column placed in a collection tube, avoiding any pellet material.
  3. Centrifuge at 13 000 rpm for 1 min to allow plasmid DNA binding to the column membrane.
  4. Discard the flow-through.

E. Wash Steps

  1. (Optional) For high-endotoxin strains, apply PB buffer wash; for standard laboratory strains, this step can be omitted.
  2. Add 750 µL PE buffer (ethanol wash) to the column and centrifuge at 13 000 rpm for 1 min.
  3. Discard the flow-through and perform a dry spin (13 000 rpm, 1–2 min) to remove residual ethanol.
  4. Transfer the column to a fresh 1.5 mL microcentrifuge tube.

F. Elution

  1. Pre-warm nuclease-free water (NFW) to 60 °C.
  2. Add 20 µL of warmed NFW to the center of the column membrane.
    Rationale: Warm water enhances elution efficiency, and a small volume yields higher plasmid concentration.
  3. Incubate at room temperature for 5 min.
  4. Centrifuge at 13 000 rpm for 5 min to collect the eluted plasmid. Label eluates appropriately (e.g., Sample A, Sample B).

G. Dilution and Quantification

  1. Prepare a 1:5 dilution by mixing 2 µL plasmid DNA with 8 µL NFW.
  2. Measure concentration and purity using a NanoDrop spectrophotometer.

NanoDrop Quantification

Principle:
NanoDrop measures absorbance of nucleic acids at 260 nm. The Beer–Lambert law is used to calculate concentration.

Procedure:

  1. Blank the instrument with NFW.
  2. Load 2 µL of sample on the pedestal.
  3. Record readings in triplicate.

Protocol: Restriction Digestion and Gel Electrophoresis

A. Restriction Digestion of Plasmid DNA

Objective:
To linearize the pET-28b(+) plasmid using BamHI and HindIII restriction enzymes for subsequent ligation.

Procedure:

  1. Reaction Setup (20 µL total):
Component
Plasmid DNA
10× CutSmart Buffer
Restriction enzymes (BamHI + HindIII)
Nuclease-free water
  1. Mixing: Briefly vortex and spin down the reaction mixture.
  2. Incubation: Incubate at 37 °C for 2 h in a thermocycler (lid heating off). Hold at 4 °C after completion.

B. Restriction Digestion of Gene Inserts (Engineered MT and ATPCS)

Objective:
To excise the engineered gene inserts (MT and ATPCS) from pTWIST Amp vectors using BamHI and HindIII.

Procedure:

  1. Reaction Setup (20 µL total per sample):
Component
Gene DNA (MT or ATPCS)
10× CutSmart Buffer
BamHI
HindIII
Nuclease-free water
  1. Mixing: Briefly vortex and centrifuge.
  2. Incubation: Incubate at 37 °C for 1.5 h, followed by a hold at 4 °C.

C. Agarose Gel Preparation (0.8%)

Objective:
To prepare a 0.8% agarose gel for resolving restriction-digested DNA samples.

Procedure:

  1. Gel composition: 0.4 g agarose in 50 mL 1× TAE buffer.
  2. Buffer preparation: 1× TAE obtained by diluting stock 5× TAE solution.
  3. Dissolution: Heat until agarose completely dissolves; compensate for evaporation with ~5 mL additional buffer.
  4. Cooling: Allow to cool to ~50–55 °C.
  5. Staining: Add SYBR Safe dye (non-carcinogenic alternative to EtBr).
  6. Casting: Pour gel into casting tray with comb, allow to solidify for 10–15 min.
  7. Running buffer: Fill gel tank with 1× TAE.

D. Sample Loading and Gel Electrophoresis

Objective:
To visualize DNA fragments resulting from restriction digestion.

Procedure:

  1. Sample preparation: Mix each digestion reaction with 4 µL DNA loading dye.
  2. Samples loaded:
    • Plasmid digestion (pET-28b(+))
    • Gene digestions (MT and ATPCS)
    • DNA ladder (molecular weight marker)
  3. Electrophoresis: Run under standard voltage and duration for resolution of DNA bands.

Protocol: Gel elution and Ligation

QIAquick Gel Extraction / PCR & Gel Cleanup Protocol (Standardized)

Starting material: Up to 10 µg DNA (70 bp – 10 kb)
Sample volume in this protocol: 50 µl gel slice or PCR product

Materials Required:

Notes Before Starting:

  1. DNA binding to the column membrane is efficient only at pH ≤7.5; Buffer QG should appear yellow.
  2. Maximum gel per spin column: 400 mg. For agarose >2%, increase Buffer QG volume 2×.
  3. All centrifugation steps are performed at 17,900 × g (13,000 rpm) unless otherwise indicated.
  4. For applications sensitive to salts (sequencing, blunt-end ligation), allow Buffer PE to stand on the column for 2–5 min before final centrifugation.

1. Gel Slice Preparation

  1. Excise the desired DNA fragment from the agarose gel using a clean scalpel.
  2. Transfer the gel slice to a colorless microcentrifuge tube.
  3. Weigh the gel slice and add 3 volumes of Buffer QG per 1 volume of gel (e.g., for 50 µl gel, add 150 µl Buffer QG). For agarose >2%, add 6 volumes.

2. Gel Dissolution

  1. Incubate the gel slice at 50 °C for 10 min, or until fully dissolved.
  2. Vortex gently every 2–3 min to facilitate dissolution.
  3. Verify the solution color: it should appear yellow. If orange or violet, add 10 µl 3 M sodium acetate (pH 5.0) and mix until the color turns yellow.

3. DNA Precipitation Enhancement

  1. Add 1 volume of 100% isopropanol to the dissolved gel solution and mix thoroughly.

4. DNA Binding to Column

  1. Place a QIAquick spin column into a 2 ml collection tube.
  2. Apply the gel/isopropanol mixture to the column.
  3. Centrifuge for 1 min. Discard flow-through and return the column to the same tube.
  4. For sample volumes >800 µl, load and centrifuge in multiple steps.

Optional: For sequencing or sensitive downstream applications, pre-wash the column by adding 500 µl Buffer QG and centrifuging for 1 min. Discard flow-through.

5. Column Wash

  1. Add 750 µl Buffer PE to the column.
  2. Centrifuge for 1 min. Discard flow-through and return the column to the same tube.
  3. Allow column to stand 2–5 min if downstream applications are salt-sensitive.
  4. Centrifuge for 1 min to remove residual wash buffer completely.

6. DNA Elution

  1. Transfer the column to a clean 1.5 ml microcentrifuge tube.
  2. Add 50 µl Buffer EB (or nuclease-free water) to the center of the column membrane.
  3. Centrifuge for 1 min to elute DNA.
  4. For higher DNA concentration:
    • Add 30 µl Buffer EB to the membrane center.
    • Let stand 1 min, then centrifuge 1 min.
    • Extend incubation up to 4 min to increase yield if necessary.

- Overnight ligation of the vector pET28b(+) with the respective genes was set up - following transformation and plating on LB+ kanamycin plates.

RESULT

Transformed AtPCS Plate Wetlab Documentation
Transformed MT Engineered Wetlab Documentation
Transformed PCS Plate Wetlab Documentation
Transformed MT Native Wetlab Documentation
pET 28(b) Wetlab Documentation
pET 28(b) Wetlab Documentation
AtPCS Wetlab Documentation
MT engg Wetlab Documentation

In summary, the genetic constructs were successfully digested and ligated into the pET28b vector. The ligation products were transformed into E. coli cells, which were subsequently plated on kanamycin-containing LB agar. Colony formation was observed, indicating successful uptake of the recombinant plasmids (images of colonies are provided below). To verify the insert presence and correct digestion, the plasmid DNA was analyzed on a 1% agarose gel, and the expected fragment sizes were observed (gel images are provided below), confirming the successful construction of the desired recombinant clones.