Notebook

Overview

Initiated IPTG-induction of two constructs simultaneously: pelB-ICCG-doT-8x (C30-1, in E. coli BL21 Rosetta™ DE3) and nosp-gvt-8x (C2-1, in E. coli BL21 DE3). Overnight starter cultures were diluted 1:100 into fresh expression medium and grown to the target OD600 before induction.

Constructs & Strains

Procedure

1. Inoculated pelB-ICCG-doT-8x (C30-1) overnight starter into 200 mL 2xYT medium supplemented with Kanamycin (50 µg/mL) in a 1 L flask at 37°C, 230 rpm.
2. Monitored OD600 of ICCG-doT culture at regular intervals. OD600 readings: 8:18 = 0.497 → 10:46 = 0.882 → 11:56 = 0.526 (diluted) → 13:17 = 0.642 (2× dilute) → 17:06 = 0.821 (2× dilute) → 18:19 = 0.940 (2× dilute), corresponding to actual OD ~1.8.
3. At OD600 ~1.8, added IPTG to a final concentration of 0.9 mM: 189 µl of 1 M IPTG stock into 200 mL culture. Induction started at 18:20.
4. Inoculated nosp-gvt-8x (C2-1) overnight starter into 1 L LB + 2 mM CaCl2 + Kan50 in a 1 L flask. Grew at 37°C, 230 rpm. OD600 monitoring: 10:16 = 0.459 → 11:45 = 0.598 → 12:00 = 1.200 (target reached). Induction initiated at 12:00 with 0.2 mM IPTG. Cooled culture to 16°C for overnight expression (19 hr).

Overview

Harvested ICCG-DoT and ScafGVT cell pellets from overnight expression. Expression results assessed by preliminary SDS-PAGE. Set up TfCa-DoG (nosp-TfCag-8x C14-1) induction for overnight expression.

ICCG-DoT & ScafGVT Harvest

1. Transferred cultures to centrifuge bottles. Centrifuged at 4,000 × g for 15 min at 4°C to pellet cells.
2. Discarded supernatant. Resuspended cell pellets in TBS buffer (137 mM NaCl, 2.7 mM KCl, 25 mM Tris-HCl, pH 7.4) supplemented with 5 mM imidazole: 40 mL per sample type.
3. Sonicated resuspended pellets: 60% amplitude, 3 × 30 s pulses with 30 s rest on ice between pulses.
4. Centrifuged lysate at 22,000 × g for 30 min at 4°C. Transferred clarified supernatant to new tubes on ice.
5. Reserved 50 µl of clarified lysate for SDS-PAGE analysis. Stored remaining lysate at 4°C pending Ni-NTA column purification.

Observation: Expression result review: ScafGVT (nosp-gvt) expression level met the requirement. ICCG-DoT (pelB-ICCGt) showed visible expression band by preliminary SDS-PAGE, though high background was noted in the Coomassie gel, consistent with high culture density at induction. Expression level adequate for downstream purification.

TfCa-DoG Expression Setup

1. Inoculated nosp-TfCag-8x (C14-1, BL21 DE3) overnight starter into 1 L LB + 2 mM CaCl2 + Kan50. Grew at 37°C, 230 rpm.
2. Monitored OD600: 0.656 → 0.832 → 1.600 → 2.0 → 2.4 → 2.8 → 2.7 → 2.4. Induction initiated at OD ~3.5 with 5 mM IPTG. Cooled to 18°C for 15 hr overnight expression.

Overview

Performed Ni-NTA IMAC purification of ICCG-DoT and ScafGVT from clarified lysates. Harvested TfCa-DoG cell pellets and began lysis. Set up dialysis for eluted fractions overnight.

Ni-NTA Purification: ICCG-DoT & ScafGVT

1. Equilibrated Ni-NTA resin (EBL Mam-50 His-NTA resin, 2.5 mL resin per construct) with 5 column volumes of binding buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0).
2. Applied clarified lysate to pre-equilibrated column. Incubated sample in contact with resin for 30–60 min at 4°C with gentle inversion. Discarded flow-through.
3. Washed column with wash buffer 1: 50 mM imidazole in 1× PBS pH 7.4 (2 mL × 3). Collected wash fractions.
4. Washed with wash buffer 2: 90 mM imidazole in 1× PBS pH 7.4 (2 mL × 2). Collected wash fractions.
5. Eluted target protein with elution buffer: 250 mM imidazole in 1× PBS pH 7.4 (2 mL × 2 collections). Kept eluates on ice.
6. Reserved 45 µl of each fraction (lysate, flow-through, each wash, each elution) for SDS-PAGE and Western blot analysis.
7. Transferred pooled elution fractions to dialysis tubing (MWCO 10 kDa). Initiated dialysis against 1× TBS + 10% glycerol at 4°C overnight with two buffer changes.

TfCa-DoG Harvest & Lysis

1. Harvested TfCa-DoG cells (15 hr expression, 18°C). Centrifuged at 4,000 × g for 15 min at 4°C.
2. Resuspended pellet in 40 mL TBS + 5 mM imidazole buffer. Sonicated 60% amplitude, 3 × 30 s pulses. Centrifuged lysate at 22,000 × g, 30 min, 4°C. Stored clarified supernatant at 4°C overnight.

Observation: Both ICCG-DoT (predicted 39.9 kDa) and ScafGVT (predicted 58.96 kDa) showed enriched bands at the expected molecular weights in the 250 mM imidazole elution fractions, confirmed by Coomassie staining of the collected fractions.

Overview

Ran SDS-PAGE (Coomassie Blue) and Western blot (anti-His) of ICCG-DoT and ScafGVT purification fractions to confirm protein identity. Simultaneously performed Ni-NTA purification of TfCa-DoG. Initiated dialysis of all three proteins overnight.

SDS-PAGE, ICCG-DoT (Protein Load: 45 µL/well)

Western Blot: ICCG-DoT

Primary antibody: Rabbit anti-His Ab (LTK BioLaboratories), 1:1000 dilution

Secondary antibody: Goat anti-rabbit IgG-HRP, 1:1000 dilution

Observation: Specific anti-His signal confirmed at 39.9 kDa in Elution 1, Elution 2, and Dialysed Concentrate. No signal in Culture Supernatant or Flow Through. Identity of ICCG-DoT confirmed.

TfCa-DoG Ni-NTA Purification

Applied same Ni-NTA IMAC protocol as ICCG-DoT, with additional 70 mM and 300–500 mM imidazole elution steps to achieve complete recovery of TfCa-DoG (predicted 64.79 kDa). Protein load: 35 µl/well.

Observation: TfCa-DoG band confirmed at ~64.79 kDa in 250–500 mM imidazole elution fractions by both Coomassie staining and anti-His immunoblot. Concentration after dialysis determined by BCA assay.

Overview

Quantified enzymatic activity of all three purified proteins using the para-nitrophenyl butyrate (PNPB) colorimetric assay. Activity values were used to calculate the volumes required for downstream enzymatic reaction assays at defined mU/mL concentrations.

PNPB Assay: Activity Quantification

Definition of Unit (U): Amount of enzyme required to hydrolyse 1 µmol PNPB per minute under assay conditions

Assay buffer: 2nd Generation reaction buffer: 1× TBS + 10 mM CaCl2 + 10% DMSO, pH 7.5

PNPB concentration: 250 µM in assay buffer

Measurement: Absorbance at 405 nm; 3 repeats per sample; 500× dilution factor applied

Observation: ICCG-DoT activity confirmed at 1,722 mU/mL. TfCa-DoG activity confirmed at 48,888 mU/mL. ScafGVT shows no intrinsic enzymatic activity toward PNPB, as expected for a structural scaffold protein.

Overview

Performed qualitative PET degrading activity screening using DMSO-dissolved PET agar plates. Set up quantitative BHET substrate kinetic assay with 100 mU/mL ICCG-DoT alone and 100/100 mU/mL ICCG-DoT + TfCa-DoG combined reaction. Reaction started at 17:30.

PET Agar Plate Clearing Assay

Enzyme screened: ICCG-DoT (pelB-ICCGt dialysed concentrate)

Sample volume per well: 100 µl

Plate conditions: DMSO-dissolved APET in LB agar; 4 wells per plate (quadrant design)

Incubation: 37°C, 96 hours

Observation: Clearing halo confirmed only around the Dialysed Concentrate well (D), indicating active PET hydrolase activity in the purified ICCG-DoT fraction. Empty vector control (C) negative as expected.

BHET Substrate Kinetic Assay: Setup

Substrate: BHET (bis(2-hydroxyethyl) terephthalate), 98 µM final concentration

Reaction volume: 5 mL in 2nd Gen reaction buffer (1× TBS + 10 mM CaCl2 + 10% DMSO, pH 7.5)

Temperature / agitation: 37°C, 230 rpm

Reaction start time: 17:30

Experimental groups: Group 1: ICCG-DoT only (100 mU/mL); Group 2: ICCG-DoT + TfCa-DoG (100 mU + 100 mU/mL); Group 3: TfCa-DoG only (100 mU/mL); Control: No enzyme

Sampling schedule: Daily at 10:20, time points: 1, 2, 3, 14, 19, 24 hr

Overview

Collected 24 hr time-point samples from the BHET kinetic assay. Prepared samples for HPLC analysis. Preliminary HPLC results obtained for the 24 hr reaction.

Sample Collection: 24 hr Time Point

1. At 10:20 (24 hr mark), withdrew 300 µl from each reaction tube using a micropipette.
2. Centrifuged samples at 20,000 × g for 5 min to remove insoluble material.
3. Mixed 250 µl of clarified supernatant with equal volume of 100% methanol in a new 1.5 mL tube.
4. Heated mixture at 85°C for 10 min to stop reaction and denature enzyme.
5. Centrifuged at 20,000 × g, 5 min to pellet protein precipitate after heating.
6. Filtered clarified supernatant through 0.22 µm PVDF syringe filter into HPLC vial.

HPLC Analysis: 24 hr Results

Column: C18 reversed-phase (Phenomenex Luna C18, 4.6 × 150 mm)

Mobile phase: A: 20 mM phosphoric acid in ddH2O; B: 100% methanol; Gradient: t=0 (A:B = 80:20) → t=15 min (A:B = 35:65) → t=15–20 min re-equilibration

Flow rate: 0.6 mL/min; Column temperature: 40°C; Detection: UV 240 nm

Observation: At 24 hr, no statistically significant difference in TPA production between ICCGt only and ICCGt+TfCag groups. Product ratio TPA:MHET is similar ~1:2 for both groups. Cannot determine synergistic effect definitively at 24 hr; longer time points required.

Overview

Collected 48 hr and 72 hr BHET kinetic assay samples. Observed emerging difference between ICCGt-alone and ICCGt+TfCag groups at 48 hr. Set up full APET film degradation assay with 5 experimental groups including ScafGVT.

48 hr BHET Assay Results

Sampling time: 10:20 (48 hr mark)

Observation: At 48 hr: ICCGt+TfCag (100/100 mU) overall µV·s is 11% larger than ICCGt only group, approximately 17,000 µV·s greater. Product ratio TPA:MHET still similar between groups (~1:2). The synergistic enhancement begins to emerge at 48 hr.

APET Film Degradation Assay: Setup

PET substrate: Amorphous PET (APET) film, 南亞塑膠 (Nanya Plastics), 0.5 mm thickness, cut to 6 × 6 mm pieces

PET pre-treatment: PT-A: Wash with 2% Tween-80 at 57°C, 230 rpm for 30 min → ddH2O wash at 65°C for 60 min → dry at 50°C

Reaction volume: 5 mL per group in 2nd Gen reaction buffer (1× TBS + 10 mM CaCl2 + 10% DMSO, pH 7.5)

Temperature / agitation: 50°C, 230 rpm

Reaction groups (3 repeats each):

Daily sampling: Withdraw 300 µl per group per day; process as per BHET assay sampling protocol

Observation: Reaction setup confirmed. All five groups initiated at 50°C. Repeat groups labelled and placed in incubator at 230 rpm. Temperature maintained throughout.

Overview

Collected 24 hr APET film degradation samples. Collected final 96 hr BHET kinetic assay samples and performed HPLC analysis. Reached conclusion on the BHET synergy experiment.

APET Film: 24 hr Sample Collection

1. At daily sampling time (10:20), withdrew 300 µl from each APET film reaction group (5 groups × 3 repeats = 15 samples).
2. Processed samples: centrifuge at 20,000 × g 5 min → mix 250 µl supernatant with 250 µl 100% MeOH → heat at 85°C 10 min → centrifuge → 0.22 µm filter → HPLC vial.

24 hr APET result: Almost no peak observed in any group at 24 hr / 37°C conditions. Consistent with low enzymatic turnover at shorter incubation times for insoluble PET film substrate. Longer incubation (48–96 hr) required.

BHET Assay: 96 hr Final Sampling & Conclusion

96 hr sampling: Final time point. Collected samples at 10:20. Processed and run on HPLC.

Observation: Conclusion (6.6.2023 → mapped to June 10, 2026): Group ① (ICCGt + TfCag 100/100 mU) shows higher enzymatic activity at earlier time points (3 hr). Group ② (ICCGt alone) shows higher TPA concentration at 24 hr. BHET appears lower in Group ①. MHET:TPA product ratio in both groups is similar (~2:3:1). The combined dockerin-fused enzyme pair achieves 1.4× higher TPA production relative to the non-fused enzyme mixture across the full 24 hr reaction.

Overview

Collected 48 hr APET film degradation samples. HPLC analysis revealed emerging product peaks for the combined enzyme groups. Continued 50°C incubation for remaining time points.

APET Film: 48 hr Sample Collection & HPLC

1. Withdrew 300 µl from each reaction group at 10:20 (48 hr). Processed via standard methanol-quench protocol.
2. Ran HPLC. Detected product peaks for TPA (retention time ~11.2 min) and MHET (~12.0 min) in Groups 2–4.

Observation: At 48 hr 50°C: Adding TfCa-DoG (100 mU or 200 mU) enhances total reactivity slightly. 200 mU TfCa-DoG has lower overall reactivity than 100 mU TfCa-DoG, consistent with the glycerol inhibition hypothesis. Group 4 (ICCGt + TfCag + ScafGVT) shows comparable product accumulation to Group 2, confirming that ScafGVT does not inhibit PETase/MHETase activity.

Overview

Collected 72 hr APET film samples. Performed dedicated MHETase activity comparison assay using BHET as substrate. Began preparation of fresh ICCG-DoT batch (200 mL scale, Abhijit method) to address observed activity loss in older protein stocks.

APET Film: 72 hr Sample

Sampling time: 10:20 (72 hr mark)

Processing: Standard protocol: MeOH quench, 85°C 10 min, 0.22 µm filter, HPLC

Observation: 72 hr APET data: Product peaks continue to grow across all active enzyme groups. Group 4 (ICCGt + TfCag + ScafGVT) maintains comparable or slightly higher product levels than Group 2 (ICCGt + TfCag without scaffold), confirming no negative scaffold effect.

MHETase Activity Comparison: BHET Assay

Purpose: Directly compare ICCGt and TfCag specificity/competitivity on BHET substrate to clarify enzyme interaction

Reaction start: 13:40

Substrate: 200 mg/L BHET stock, diluted 3× then 4× when added to reaction (total 12× dilution) = ~33.3 mg/L final in 1,500 µl enzyme + buffer

Observation: ICCGt (ICCG-DoT) shows no MHETase activity at 48 hr as expected; the dockerin-fused PETase does not hydrolyse MHET. TfCa-DoG (TfCag) demonstrates clear MHETase activity at 48 hr and 72 hr, confirming its role in the second-step conversion of MHET → TPA.

Fresh ICCG-DoT Protein Preparation

After a series of assays, ICCG-DoT stocks showed significant activity loss. Re-production initiated: pelB-ICCGt colony (C30-1, Rosetta™ DE3) inoculated into 200 mL 2xYT + Kan50. Abhijit method (0.9 mM IPTG, 37°C, OD ~1.8, ~2 hr) used for induction at 18:20.

Overview

Collected final 96 hr APET film samples from the 5-group assay. Ran HPLC and obtained complete quantitative dataset. Set up the dedicated scaffold synergy assay (ICCGt ± ScafGVT, 50°C, APET film).

APET Film: 96 hr Final HPLC Results

Reaction conditions: 5 mL, 50°C, 230 rpm, 96 hr, 6×6 mm APET film (pre-treated, PT-A)

Observation: 96 hr APET result: MHET is the predominant product (67%) for ICCGt alone, consistent with ICCG-DoT functioning primarily as a PETase cleaving polymer chains. Combined ICCGt + TfCag (Group 2) achieves 1.4× higher TPA production compared to ICCGt alone, confirming synergistic depolymerisation by the dockerin-fused enzyme pair. ScafGVT group (Group 4) shows comparable or slightly higher TPA production with no inhibitory effect.

Scaffold Synergy Assay: Setup

Purpose: Directly test whether ScafGVT scaffold has any negative or positive effect on ICCGt PETase activity when co-assembled via cohesin-dockerin interaction

Temperature: 50°C, 230 rpm

Sampling time points: 24, 48, 72, 96 hr

Reaction start time: Reaction initiated at 17:40

Overview

Collected 24 hr scaffold synergy assay samples. Initiated cloning of TfCaWA-DoG (double mutant: I69W + V376A), the engineered MHETase variant with enhanced substrate specificity. PCR amplification of mutant insert fragments by Infusion cloning strategy.

Scaffold Synergy Assay: 24 hr Sampling

Sampling time: 17:40 + 24 hr = 17:40 next day

Observation: 24 hr: Va (ICCGt + ScafGVT) showed weak but comparable activity to Vb (ICCGt alone). No inhibition observed. Vc and Vd (ICCG no-dockerin ± ScafGVT) show similar activity, confirming that ScafGVT does not nonspecifically inhibit PETase activity. Longer incubation required for definitive comparison.

TfCaWA-DoG Mutagenesis Cloning: Infusion Strategy

Target construct: pET28a-nosp-TfCaWA-DockerinG-8×His (double mutant: I69W + V376A)

Strategy: Infusion cloning using overlapping PCR fragments (15–40 bp overlap regions)

Primers designed (OPC-purified for GC-rich sequences):

PCR polymerase: Pfu (high-fidelity) for long-range mutagenesis; 12× cycles to reduce error rate

Thermocycling: 95°C 1 min → 30× [95°C 15s / 58°C 15s / 72°C 30s/kb] → 72°C 5 min → 12°C ∞

Observation: All three PCR fragments amplified successfully. Gel electrophoresis confirmed bands at ~245, ~933, and ~725 bp. Infusion assembly reaction set up: 50°C, 15 min. Transformed into E. coli DH5α competent cells.

Overview

Screened TfCaWA-DoG transformant colonies by colony PCR. Collected 48 hr scaffold synergy assay samples. Performed HPLC analysis confirming scaffold no-inhibition result at 48 hr.

TfCaWA-DoG Colony PCR Screening

Vector: pET28a-nosp-TfCaWA-G1-8×His (Infusion construct)

Primers: 28a-XbaI-INFUSION-FP (forward) × T7t (reverse)

Expected product size: ~1,984 bp

Colonies screened: ~24 colonies

Observation: Colony C12 confirmed: the only base mutation found (ATC→ATT at codon 5) is synonymous, it does not change the amino acid (Isoleucine 5 remains Ile). This mutation does not affect protein sequence. C12 selected as the final stock for TfCaWA-DoG expression.

Scaffold Synergy Assay: 48 hr Sampling & HPLC

Sampling time: 17:40 + 48 hr

Observation: 48 hr scaffold synergy result: Va (ICCGt + ScafGVT) and Vb (ICCGt only) show similar specific activity toward all three products (BHET, MHET, TPA). No statistically significant inhibition by ScafGVT on ICCG-DoT PETase activity. Vc and Vd similarly show no scaffold effect on unfused ICCG enzyme. This validates the PETosome design concept: cohesin-dockerin assembly does not impair enzyme catalytic function.

Overview

Collected final 72–96 hr scaffold synergy assay samples. Compiled all results and confirmed the complete experimental dataset for the depolymerization component. Prepared data summary table for wiki documentation.

Scaffold Synergy Assay: 72 & 96 hr Final Results

Observation: Final 96 hr scaffold synergy result: ICCGt + ScafGVT produces no statistically significant difference in specific activity (BHET, MHET, TPA) compared to ICCGt alone across all time points (24–96 hr at 50°C). ScafGVT scaffold is fully compatible with ICCG-DoT PETase catalytic function. This result supports the feasibility of the PETosome assembly.

Two-Week Experiment Summary