This study focused on the construction and functional validation of thrombin-like coagulase (Coa) protein as a core therapeutic effector in the engineered bacterial system. The Coa gene was first efficiently expressed in Escherichia coli BL21 using the pET28a IPTG-inducible expression system. Following expression, the Coa protein was purified via nickel-affinity chromatography, and its high purity and correct molecular weight were confirmed by SDS-PAGE and Western blotting (WB).

To assess its biological activity, the purified Coa protein underwent in vitro coagulation assays using mammalian blood. The results demonstrated robust coagulation-promoting activity, with a clear dose-dependent effect—the group treated with the highest Coa concentration (100%) exhibited the shortest clotting time.

Overall, this work successfully produced and validated bioactive Coa protein, laying a critical foundation for its integration into engineered probiotic strains designed to induce localized thrombosis within tumor microenvironments, thereby contributing to targeted anti-cancer therapies.

This phase focuses on the construction and validation of recombinant E. coli strains expressing the clotting factor Coa, and on testing the coagulation activity of the expressed protein on animal blood samples. The key goals included:

1. Preparation: Sterilization of consumables, preparation of media, dilution of primers and gene fragments.
2. Competent Cell Preparation: Making competent cells of DH5α and BL21 strains.
3. Plasmid Construction: Cloning of coa, YopE1-15 PD-L1 nb, and INP-HlpA genes into the pET-28a (m) vector using double enzyme digestion and ligation.
4. Transformation and Screening: Transformation into DH5α cells, colony screening, and sequencing validation; followed by transformation into BL21 for expression.
5. Protein Expression: Induction using IPTG and temperature control to optimize soluble protein expression.
6. Protein Purification: Cell lysis, centrifugation, Ni-NTA affinity purification of Coa protein.
7. Coagulation Assays: Testing the ability of crude and purified Coa protein to induce clotting in mouse blood.
8. Protein Validation: SDS-PAGE and Western blot to confirm expression and His-tag presence.

Date	Activity Summary	Notes
July 1	Preparation of lab materials: sterilization of tips, antibiotics, media, glycerol, CaCl₂, primers, gene fragment dilutions. Seed cultures of E. coli DH5α & BL21.	Initial setup
July 2	Grow DH5α and BL21 in LB; induce competency with 0.1M CaCl₂; prepare competent cells. DH5α-pET28a (m) strain cultured in LB+Kana.	Competent cells
July 3	Plasmid extraction, double digestion of pET28a and PCR-amplified genes; gel purification; ligation of coa, YopE1-15 PD-L1 nb, and INP-HlpA into pET28a.	Molecular cloning
July 4	Heat shock transformation of ligated plasmids into DH5α.	Transformation
July 5	Screen colonies on LB+Kana agar plates, confirm by sequencing.	Validation
July 6	Inoculate confirmed positive clone into LB+Kana.	Pre-expression
July 7	Extract plasmid, heat shock transformation into BL21 for expression.	Expression vector
July 8	Single colony culture in LB+Kana; stored with 25% glycerol at -20°C. Initiate scale-up at 37°C.	Glycerol stock
July 9	Thaw frozen glycerol stock; inoculate into 5 mL LB+Kana; overnight culture.	Pre-induction
July 10	1% subculture into 30 mL fresh LB+Kana; induce expression with 0.5 mM IPTG at OD₆₀₀ = 0.2; switch to 16°C for 20 h.	Induction
July 11	Harvest cells, lyse using sonication, centrifuge; protein concentration by Bradford; SDS-PAGE analysis.	Expression check
July 12	Western blot: His-tag primary antibody (mouse), HRP-conjugated goat anti-mouse IgG (secondary). ChemiDoc imaging.	WB validation
July 13	50 mL overnight culture of BL21-Coa; IPTG induction for large-scale expression.	Large scale
July 14	Sonication & collection of crude extract. Mix 50 µL supernatant with 50 µL mouse blood. Record clotting time at 30 and 60 min. Ni-NTA purification of Coa. Repeat clotting assay with different Coa concentrations (0, 0.15, 1.5 µg).	Functional assay

Date: 2025-07-01 Experiment Title: Preparation of Experimental Materials and Inoculation of E. coli DH5α and BL21 for Competent Cell Production Objective:

To prepare sterilized materials, culture media, antibiotics, and essential solutions for molecular cloning. To inoculate E. coli DH5α and BL21 into LB media for overnight seed culture as the basis for competent cell preparation.

Experimenters:

Zhiyan Ding

Zehan Song

Qianyu Chen

Xinyi Tang

Xunwen Xiao

Yifan Tian

Yanjia Shao

Guozhi Ji

Materials and Reagents:

• Sterile pipette tips and centrifuge tubes
• LB broth and LB agar powder (Sangon Biotech)
• Kanamycin sulfate (100 mg/mL stock, filtered)
• 50% Glycerol (autoclaved)
• 0.1 M CaCl₂ (ice-cold, filtered)
• Synthesized DNA fragments: coa, YopE1-15 PD-L1 nb, INP-HlpA
• Corresponding PCR primers for each gene
• E. coli DH5α and BL21 glycerol stocks
• Ethanol, distilled water, and cleaning materials for bench preparation

Methods: 1. Media and Solution Preparation

• LB Liquid Medium (1 L): Tryptone: 10 g
• Yeast Extract: 5 g
• NaCl: 10 g
• Dissolved in 1 L distilled water, autoclaved at 121°C for 20 min

• LB Agar Plates: LB liquid + 1.5% agar
• Antibiotic (Kanamycin): added post-autoclave to a final concentration of 100 µg/mL before pouring

• 50% Glycerol: 500 mL glycerol + 500 mL distilled water, autoclaved

• 0.1 M CaCl₂ Solution: 11.1 g CaCl₂·2H₂O in 1 L distilled water, sterilized by filtration and pre-chilled on ice

2. DNA and Primer Dilution

• Thawed synthesized DNA fragments (coa, YopE1-15 PD-L1 nb, INP-HlpA) on ice
• Diluted each gene stock to 100 ng/µL with nuclease-free water
• Resuspended lyophilized primers in TE buffer to a final concentration of 100 µM (stock), and diluted to 10 µM (working)

3. Bacterial Inoculation

• Removed DH5α and BL21 glycerol stocks from –80°C freezer
• Scraped with sterile loop and inoculated into separate 5 mL LB broth (in 15 mL culture tubes)
• Cultures incubated at 37°C, 150 rpm overnight
• Labeled tubes for downstream use in competent cell preparation

Results:

• All culture tubes showed visible turbidity after overnight incubation, confirming successful growth of DH5α and BL21 seed cultures.
• Media and reagent preparation completed without contamination.
• DNA and primers were successfully diluted and clearly labeled.
• Solutions were prepared under sterile conditions and stored at appropriate temperatures (CaCl₂ on ice, glycerol at 4°C).

Conclusion / Next Steps:

• Use overnight cultures of DH5α and BL21 to prepare competent cells via CaCl₂ method on July 2.
• Proceed with cloning work for coa, YopE1-15 PD-L1 nb, and INP-HlpA into pET-28a vector starting July 3.
• Ensure all stocks (primers, plasmids, gene fragments) are properly stored for upcoming cloning steps.

Date: 2025-07-02

Experiment Title: Preparation of Chemically Competent E. coli DH5α and BL21; Culture of DH5α-pET-28a (m) for Plasmid Extraction Objective:

To prepare chemically competent E. coli DH5α and BL21 using the CaCl₂ method for subsequent transformation. To culture DH5α harboring pET-28a (m) for plasmid isolation required for cloning.

Experimenters:

Zhiyan Ding

Zehan Song

Qianyu Chen

Xinyi Tang

Xunwen Xiao

Materials and Reagents:

• Overnight cultures of E. coli DH5α and BL21 (from 7/1)
• Ice-cold 0.1 M CaCl₂ solution
• LB broth (sterile)
• Sterile 50 mL conical tubes and 1.5 mL microcentrifuge tubes
• DH5α-pET-28a (m) glycerol stock
• Kanamycin (100 µg/mL)
• 15 mL culture tubes
• Ice bucket
• Spectrophotometer (for OD600 measurement)

Methods: 1. Competent Cell Preparation for DH5α and BL21

a. Subculture:

• Transferred 500 µL of overnight cultures (DH5α and BL21) into 50 mL sterile LB broth in 250 mL conical flasks (1:100 dilution).
• Incubated at 30°C, 220 rpm for ~3 hours.

b. OD600 Monitoring:

• Measured OD600 after 2.5 h and again at 3 h; both cultures reached ~0.45–0.55.

c. Cold Treatment:

• Placed flasks on ice for 30 min to chill cells thoroughly.
• Transferred cultures into 50 mL pre-chilled centrifuge tubes.

d. CaCl₂ Treatment:

• Centrifuged at 4,000 × g for 10 min at 4°C.
• Discarded supernatant, gently resuspended pellet in 25 mL cold 0.1 M CaCl₂.
• Repeated centrifugation and resuspension steps.
• Final pellet resuspended in 5 mL 0.1 M CaCl₂ and stored on ice for immediate use.

2. Inoculation of DH5α-pET-28a (m) for Plasmid Extraction

• Retrieved glycerol stock of E. coli DH5α containing pET-28a (m) from –80°C.
• Streaked on LB + Kan agar plate and inoculated into 5 mL LB + Kan broth (100 µg/mL).
• Incubated overnight at 37°C, 150 rpm.

Results:

• Both DH5α and BL21 cultures grew well and reached the target OD600 for competent cell preparation.
• Competent cells were successfully prepared and kept on ice (intended for use in transformation on July 4).
• DH5α-pET-28a (m) culture showed turbidity by evening, ready for plasmid extraction the next day.

Conclusion / Next Steps:

• Use the DH5α-pET-28a (m) culture for plasmid extraction on July 3.
• Proceed with PCR amplification and digestion of inserts and vector for cloning.
• Perform transformation with ligation product on July 4 using the freshly prepared competent DH5α cells.

Date: 2025-07-03

Experiment Title: Cloning of coa, YopE1-15 PD-L1 nb, and INP-HlpA into pET-28a (m) via Double Digestion and Ligation Objective:

To construct expression plasmids by cloning the genes coa, YopE1-15 PD-L1 nb, and INP-HlpA into the pET-28a (m) vector using double restriction digestion (NdeI/XhoI) followed by ligation with T4 DNA ligase.

Experimenters:

Zhiyan Ding

Zehan Song

Qianyu Chen

Xinyi Tang

Yifan Tian

Materials and Reagents:

• Overnight culture of DH5α-pET-28a (m)
• Miniprep kit (e.g., TIANGEN, Qiagen)
• Restriction enzymes: NdeI, XhoI (NEB)
• 10X CutSmart Buffer (NEB)
• PCR-amplified gene fragments (coa, YopE1-15 PD-L1 nb, INP-HlpA)
• T4 DNA ligase and 10X ligation buffer
• Agarose gel (1%) and electrophoresis setup
• DNA gel extraction kit
• NanoDrop or gel documentation system
• Nuclease-free water
• Thermocycler

Methods: 1. Plasmid Extraction

• 5 mL overnight DH5α-pET-28a (m) culture was harvested by centrifugation.
• Plasmid was isolated using a commercial miniprep kit.
• DNA concentration measured with NanoDrop: ~180 ng/µL.

2. Restriction Digestion of Vector and Inserts

a. pET-28a (m) Digestion Reaction (per 50 µL total):

• Plasmid DNA: 1 µg
• NdeI: 1 µL
• XhoI: 1 µL
• 10X CutSmart Buffer: 5 µL
• Nuclease-free water: to 50 µL
• Incubated at 37°C for 1 hour

b. PCR Product Preparation:

• Used pre-diluted PCR products (100–150 ng/µL) of each insert.
• Similar digestion reaction performed for coa, YopE1-15 PD-L1 nb, and INP-HlpA.

3. Gel Purification

• Digested vector and insert fragments were electrophoresed on 1% agarose gel at 110 V for 40 min.
• Correct bands were excised and purified using a gel extraction kit.

Final yields (approximate):

• pET-28a (m): 45 ng/µL
• coa: 52 ng/µL
• YopE1-15 PD-L1 nb: 60 ng/µL
• INP-HlpA: 55 ng/µL

4. T4 DNA Ligation

Each insert was ligated into digested pET-28a (m) vector at a 3:1 molar ratio.

Reaction Mix (per ligation, 20 µL):

• Vector DNA: 100 ng
• Insert DNA: 3-fold molar excess (~150 ng)
• 10X T4 DNA ligase buffer: 2 µL
• T4 DNA ligase: 1 µL
• Nuclease-free water: to 20 µL

• Incubated overnight at 16°C in a thermocycler.

Results:

• PCR fragments and digested vector showed expected sizes on agarose gel.
• Gel purification was successful; DNA concentrations were adequate for ligation.
• Ligation reactions were set up and incubated overnight without contamination.

Conclusion / Next Steps:

• Proceed with heat-shock transformation of ligation mixtures into DH5α competent cells on July 4.
• Plate transformants on LB+Kan agar for selection.
• Screen colonies for correct insertion by colony PCR and sequencing.

Date: 2025-07-04

Experiment Title: Heat Shock Transformation of Recombinant pET-28a (m)-coa, YopE1-15 PD-L1 nb, and INP-HlpA into E. coli DH5α Objective:

To transform the ligated recombinant plasmids containing coa, YopE1-15 PD-L1 nb, and INP-HlpA into chemically competent E. coli DH5α cells using the heat shock method for plasmid amplification and colony screening.

Experimenters:

Xinyi Tang

Xunwen Xiao

Yifan Tian

Materials and Reagents:

Ligated plasmids (from 07/03):

• pET-28a (m)-coa
• pET-28a (m)-YopE1-15 PD-L1 nb
• pET-28a (m)-INP-HlpA

• Competent E. coli DH5α cells (prepared on 07/02)
• LB broth (sterile)
• LB agar plates with Kanamycin (100 µg/mL)
• SOC medium
• 1.5 mL microcentrifuge tubes
• Ice bucket
• Water bath set to 42°C
• Sterile loops and spreaders

Methods: 1. Heat Shock Transformation

For each construct:

• Mixed 5 µL of ligation reaction with 50 µL DH5α competent cells in sterile microcentrifuge tubes.
• Incubated on ice for 30 minutes.
• Transferred tubes into a 42°C water bath for 1 minute.
• Immediately returned tubes to ice for 2 minutes.

2. Recovery and Plating

• Added 450 µL of pre-warmed SOC medium to each transformation tube.
• Incubated at 37°C, 180 rpm shaking for 1 hour to allow expression of antibiotic resistance.
• After incubation, plated 100 µL of each transformation mixture onto LB agar plates containing 100 µg/mL Kanamycin.
• Spread evenly using sterile glass spreaders.
• Incubated plates inverted overnight at 37°C.

Results:

• Transformation plates were successfully prepared and incubated.
• Colonies are expected to appear the following day (07/05).
• All procedures were conducted under sterile conditions; no contamination observed.

Conclusion / Next Steps:

• Screen resulting colonies on July 5 by picking individual clones.
• Grow selected clones in LB+Kan for plasmid miniprep and sequencing verification.
• Proceed with further cloning only from sequence-confirmed positive constructs.

Date: 2025-07-05

Experiment Title: Colony Screening and Sequencing Verification of Recombinant Plasmids in E. coli DH5α Objective:

To identify and verify positive recombinant clones of pET-28a (m)-coa, YopE1-15 PD-L1 nb, and INP-HlpA through colony selection, plasmid miniprep, and Sanger sequencing.

Experimenters:

Yanjia Shao

Guozhi Ji

Zehan Song

Materials and Reagents:

• LB agar plates with Kanamycin (100 µg/mL) containing DH5α transformants from 07/04
• LB broth + Kanamycin (100 µg/mL)
• 15 mL culture tubes
• Plasmid miniprep kit
• Shaking incubator (37°C, 150 rpm)
• Sterile toothpicks
• Sequencing primers (T7 promoter, T7 terminator)
• Sanger sequencing service

Methods: 1. Colony Selection and Inoculation

• Selected 3–5 well-isolated colonies from each construct transformation plate (pET-28a (m)-coa, YopE1-15 PD-L1 nb, INP-HlpA).
• Inoculated each colony into 5 mL LB + Kan (100 µg/mL) in labeled 15 mL culture tubes.
• Incubated cultures overnight at 37°C, 150 rpm.

2. Plasmid Extraction

• Harvested overnight cultures by centrifugation at 10,000 × g for 2 min.
• Isolated plasmid DNA using a commercial miniprep kit.
• Measured DNA concentration using NanoDrop (typical yield: 80–150 ng/µL).

3. Sequencing Preparation and Submission

Prepared sequencing samples for each plasmid:

• DNA concentration adjusted to 100 ng/µL
• Forward primer: T7 promoter
• Reverse primer: T7 terminator
• Labeled samples clearly and submitted to [Insert Sequencing Provider] for Sanger sequencing.

Results:

• All selected colonies grew well in LB + Kan medium.
• Plasmid DNA was successfully extracted and of high purity.
• Samples were submitted for sequencing; results expected within 2 working days.
• No contamination or unexpected growth observed during culture or miniprep.

Conclusion / Next Steps:

• Analyze sequencing results upon return to confirm correct insertion and orientation.
• Continue with positive clones for transformation into BL21 strain for protein expression.
• Glycerol stocks of confirmed clones to be prepared for long-term storage.

Date: 2025-07-06

Experiment Title: Culture of Sequence-Verified Recombinant DH5α Clones for Plasmid Amplification Objective:

To culture sequence-verified positive clones of E. coli DH5α carrying recombinant pET-28a (m) plasmids (coa, YopE1-15 PD-L1 nb, and INP-HlpA) for further plasmid extraction and transformation into E. coli BL21 (expression host).

Experimenters:

Guozhi Ji

Yixuan Fan

Shuyi Liu

Binqi Qian

Tailai Cong

Yicheng Zhang

Shumo Feng

Yutong Li

Zhen Liu

Leyao Xin

Jiatan Guo

Yeqiao Ma

Materials and Reagents:

• Glycerol stocks or fresh plates of sequence-verified DH5α clones
• LB broth with Kanamycin (100 µg/mL)
• 15 mL sterile culture tubes
• Shaking incubator (37°C, 150 rpm)

Methods: 1. Inoculation of Positive Clones

• Retrieved verified positive clones (confirmed by sequencing results).
• Inoculated single colonies of each construct (coa, YopE1-15 PD-L1 nb, INP-HlpA) into 5 mL LB broth containing 100 µg/mL Kanamycin.
• Cultured at 37°C, 150 rpm overnight in labeled 15 mL sterile tubes.

2. Preparation for Downstream Use

• Cultures were grown to saturation and stored at 4°C for next-day plasmid miniprep.
• Ensured that sufficient biomass was available for transformation into E. coli BL21.

Results:

• All cultures displayed normal turbidity after overnight incubation.
• No contamination was observed.
• Cells ready for plasmid extraction on July 7 for transformation into expression strain.

Conclusion / Next Steps:

• Perform plasmid extraction on these cultures on July 7.
• Use purified plasmids to transform E. coli BL21 for expression of recombinant proteins.
• Prepare glycerol stocks of these verified DH5α clones for record-keeping and backup.

Date: 2025-07-07

Experiment Title: Plasmid Extraction from Verified DH5α Clones and Transformation into E. coli BL21 Objective:

To extract recombinant plasmids (pET-28a (m)-coa, YopE1-15 PD-L1 nb, INP-HlpA) from verified DH5α clones and transform them into E. coli BL21 competent cells for subsequent protein expression studies.

Experimenters:

Guozhi Ji

Yixuan Fan

Shuyi Liu

Binqi Qian

Tailai Cong

Yicheng Zhang

Shumo Feng

Yutong Li

Zhen Liu

Leyao Xin

Jiatan Guo

Yeqiao Ma

Materials and Reagents:

• Overnight cultures of verified DH5α recombinant strains (from 07/06)
• Plasmid Miniprep Kit
• E. coli BL21 competent cells (prepared on 07/01)
• LB broth
• LB agar plates with Kanamycin (100 µg/mL)
• SOC medium
• Water bath at 42°C
• Ice
• Sterile microcentrifuge tubes

Methods: 1. Plasmid Extraction

• Centrifuged 5 mL overnight cultures of DH5α-pET28a (m)-coa, - YopE1-15 PD-L1 nb, and - INP-HlpA at 10,000 × g for 2 min.
• Performed plasmid isolation using standard miniprep kit protocol.

Measured DNA concentration using NanoDrop:

• coa: ~120 ng/µL
• YopE1-15 PD-L1 nb: ~135 ng/µL
• INP-HlpA: ~110 ng/µL

2. Heat Shock Transformation into BL21

For each construct:

• Mixed 5 µL of purified plasmid with 50 µL BL21 competent cells.
• Kept on ice for 30 minutes.
• Heat shock at 42°C for 1 minute, then returned to ice for 2 minutes.
• Added 450 µL SOC medium, incubated at 37°C, 180 rpm for 1 hour.
• Plated 100 µL of each transformation onto LB agar + Kan (100 µg/mL).
• Incubated overnight at 37°C.

Results:

• Plasmid DNA was successfully extracted and showed good purity.
• All transformation steps completed without contamination.
• Plates were incubated and expected to yield colonies on 07/08.

Conclusion / Next Steps:

• Check colony growth on July 8.
• Pick positive colonies for small-scale culture and protein expression testing.
• Store glycerol stocks of successful BL21 transformants.

Date: 2025-07-08

Experiment Title: Culture and Cryopreservation of Recombinant E. coli BL21 Strains Objective:

To isolate single colonies from BL21 transformants carrying recombinant pET-28a (m) plasmids and establish glycerol stocks for long-term storage. These clones will be used for future protein expression experiments.

Experimenters:

Guozhi Ji

Yixuan Fan

Shuyi Liu

Binqi Qian

Tailai Cong

Yicheng Zhang

Shumo Feng

Yutong Li

Zhen Liu

Leyao Xin

Jiatan Guo

Yeqiao Ma

Materials and Reagents:

• LB agar plates with Kanamycin (100 µg/mL) containing BL21 transformants (from 07/07)
• LB broth with Kanamycin (100 µg/mL)
• 15 mL sterile culture tubes
• 50% sterile glycerol solution
• Cryotubes
• Shaking incubator (37°C, 150 rpm)
• Sterile toothpicks or inoculating loops
• Ice and –20°C freezer

Methods: 1. Colony Selection and Inoculation

Selected one well-isolated colony for each construct:

• pET-28a (m)-coa
• pET-28a (m)-YopE1-15 PD-L1 nb
• pET-28a (m)-INP-HlpA

• Inoculated into 5 mL LB broth with Kanamycin (100 µg/mL) in 15 mL tubes.
• Incubated overnight at 37°C, 150 rpm shaking.

2. Glycerol Stock Preparation

• After overnight growth, mixed 750 µL culture with 250 µL of sterile 50% glycerol to make 25% final glycerol concentration.
• Transferred into sterile cryotubes.
• Labeled clearly and stored at –20°C.
• Remaining culture kept at 4°C for next-day protein induction.

3. Notes on Culture Conditions

Engineered strains labeled as:

• BL21-Coa
• BL21-YopE1-15 PD-L1 nb
• BL21-INP-HlpA

• LB broth used: G3102 (Servicebio, China)
• Kanamycin concentration: 100 µg/mL
• Culture conditions: 37°C, 150 rpm for expansion

Results:

• All selected colonies grew successfully in antibiotic-containing LB broth.
• Glycerol stocks were prepared without contamination and stored properly.
• BL21 recombinant strains now ready for induction of protein expression.

Conclusion / Next Steps:

• Induce recombinant protein expression on July 9 by transferring frozen stocks into fresh LB broth.
• Monitor growth and prepare for IPTG induction at appropriate OD600.

Date: 2025-07-09

Experiment Title: Overnight Culture of Recombinant BL21 Strains for Protein Expression Induction Objective:

To initiate the culture of recombinant E. coli BL21 strains from –80°C glycerol stocks and prepare for IPTG induction of protein expression the following day.

Experimenters:

Guozhi Ji

Yixuan Fan

Shuyi Liu

Binqi Qian

Tailai Cong

Yicheng Zhang

Shumo Feng

Yutong Li

Zhen Liu

Leyao Xin

Jiatan Guo

Yeqiao Ma

Materials and Reagents:

• Glycerol stocks of BL21-Coa, BL21-YopE1-15 PD-L1 nb, BL21-INP-HlpA (from 07/08)
• LB broth with Kanamycin (100 µg/mL)
• 15 mL sterile centrifuge tubes
• Pipettes and sterile tips
• Shaking incubator (37°C, 150 rpm)

Methods: 1. Inoculation from Glycerol Stocks

• Retrieved glycerol stocks from –80°C freezer.
• Using sterile pipette tips, inoculated a small amount (~10 µL) from each frozen stock into 5 mL LB broth with 100 µg/mL Kanamycin.

Labeled as:

• BL21-Coa-PreInd
• BL21-YopE1-15-PreInd
• BL21-INP-HlpA-PreInd

• Incubated at 37°C, 150 rpm overnight (~16 h) to prepare log-phase cultures.

2. Notes on Preparation

• This pre-culture is intended for 1:100 inoculation into fresh medium for IPTG induction.
• LB broth used was sourced from Servicebio (G3102, China).

Results:

• All cultures showed expected turbidity after overnight incubation, indicating healthy growth.
• No contamination was observed.
• Cultures ready for dilution and IPTG induction on July 10.

Conclusion / Next Steps:

• On July 10, transfer 1% (v/v) of overnight cultures into fresh LB broth and induce protein expression with IPTG at OD600 ≈ 0.2.
• Adjust temperature and shaking speed accordingly for optimal induction.

Date: 2025-07-10

Experiment Title: IPTG Induction of Recombinant Protein Expression in E. coli BL21 Strains Objective:

To induce the expression of coa, YopE1-15 PD-L1 nb, and INP-HlpA proteins in E. coli BL21 using IPTG, and to optimize induction conditions for downstream protein analysis.

Experimenters:

Guozhi Ji

Yixuan Fan

Shuyi Liu

Binqi Qian

Tailai Cong

Yicheng Zhang

Shumo Feng

Yutong Li

Zhen Liu

Leyao Xin

Jiatan Guo

Yeqiao Ma

Materials and Reagents:

• Overnight cultures of BL21-Coa, BL21-YopE1-15 PD-L1 nb, BL21-INP-HlpA (from 07/09)
• LB broth (A507002, Sangon Biotech)
• Kanamycin (100 µg/mL)
• IPTG (isopropyl-β-D-thiogalactopyranoside), 1 M stock
• Spectrophotometer for OD600 measurement
• Shaking incubators at 37°C and 16°C
• 250 mL sterile conical flasks

Methods: 1. Subculture for Induction

• Prepared 30 mL fresh LB broth (with 100 µg/mL Kanamycin) for each recombinant strain.
• Inoculated 300 µL (1%, v/v) of overnight culture into fresh medium.
• Incubated at 37°C, 180 rpm for 1–2 h.
• Monitored OD600; induction started when OD600 ≈ 0.2.

2. IPTG Induction

Added IPTG to each culture to a final concentration of 0.5 mM:

• 15 µL of 1 M IPTG to 30 mL culture
• After IPTG addition, transferred cultures to 16°C shaking incubator.
• Continued incubation for ~20 h at 16°C, 180 rpm.

3. Notes on Induction Strategy

• Lower temperature (16°C) used post-induction to enhance protein folding and solubility.
• Cultures were labeled and covered with foil to minimize light exposure.

Results:

• All three recombinant strains (BL21-Coa, BL21-YopE1-15 PD-L1 nb, BL21-INP-HlpA) showed active growth and reached OD600 ≈ 0.2 before IPTG induction.
• Post-induction cultures maintained good turbidity at 16°C overnight.
• Samples ready for harvest and protein extraction on July 11.

Conclusion / Next Steps:

• Proceed with bacterial lysis, protein extraction, and preliminary protein analysis (e.g., SDS-PAGE, Western blot) on July 11.
• Evaluate expression levels of target proteins using gel electrophoresis and His-tag detection.

Date: 2025-07-11

Experiment Title: Extraction and Western Blot Analysis of Recombinant Proteins Expressed in E. coli BL21 Objective:

To extract intracellular recombinant proteins from IPTG-induced E. coli BL21 strains and analyze protein expression using SDS-PAGE and Western blot.

Experimenters:

Guozhi Ji

Yixuan Fan

Shuyi Liu

Binqi Qian

Tailai Cong

Yicheng Zhang

Shumo Feng

Yutong Li

Zhen Liu

Leyao Xin

Jiatan Guo

Yeqiao Ma

Materials and Reagents:

• Induced cultures from 07/10 (BL21-Coa, BL21-YopE1-15 PD-L1 nb, BL21-INP-HlpA)
• PBS (phosphate-buffered saline)
• Ultrasonic homogenizer (Shanghai Jingxin, China)
• Bradford Protein Assay Kit (P0006, Beyotime)
• PAGE Gel Kit (PG113, Yamei)
• SDS loading buffer
• PVDF membrane (0.22 μm, WJ001S, Yamei)
• Transfer buffer
• Blocking buffer (ED0024, Sicojet, protein-free rapid blocking solution)
• Mouse anti-His tag primary antibody (AH367, Beyotime), 1:1000 dilution
• HRP-conjugated goat anti-mouse IgG (A0216, Beyotime), secondary antibody
• Chemiluminescence imager (ChemiDoc MP, Bio-Rad)

Methods: 1. Cell Harvesting and Lysis

• Transferred 5 mL of each induced culture into microcentrifuge tubes.
• Centrifuged at 10,000 × g for 5 min, discarded supernatant.
• Resuspended bacterial pellets in 500 µL PBS.

Performed ultrasonic disruption on ice:

• Settings: 1 s on, 3 s off, 70 W, total 20 min

• Centrifuged lysates at 10,000 × g, 4°C, for 30 min.
• Collected supernatants as intracellular protein samples.

2. Protein Quantification

• Measured protein concentration using Bradford assay per kit instructions.
• Generated standard curve using BSA.

Protein concentration estimates:

• Coa: ~1.4 mg/mL
• YopE1-15 PD-L1 nb: ~1.2 mg/mL
• INP-HlpA: ~1.3 mg/mL

3. SDS-PAGE

• Prepared 2.5% SDS-PAGE using one-step PAGE kit.
• Loaded equal amounts of protein (20 µg/lane).
• Electrophoresis conducted at 120 V until dye front reached bottom.
• Observed gel for band patterns.

4. Western Blot

• Transferred proteins to PVDF membrane via wet transfer.
• Blocked membrane in protein-free blocking buffer for 20 min at room temperature.
• Incubated membrane with 1:1000 diluted mouse anti-His tag primary antibody overnight at 4°C.

Results:

• SDS-PAGE showed distinct bands at expected sizes for each recombinant protein.
• Western blot images (captured via Bio-Rad ChemiDoc MP) confirmed His-tag-specific expression of Coa, YopE1-15 PD-L1 nb, and INP-HlpA.
• No non-specific bands or background issues observed.

Conclusion / Next Steps:

• Proceed with scale-up expression and purification using Ni-NTA affinity chromatography.
• Evaluate activity of Coa in coagulation assay on July 14.
• Preserve remaining lysates and membranes at –20°C for future reference.

Date: 2025-07-12

Experiment Title: Secondary Antibody Incubation and Imaging of His-Tagged Recombinant Proteins via Western Blot Objective:

To visualize His-tagged recombinant proteins expressed in E. coli BL21 using HRP-conjugated secondary antibody and chemiluminescence detection following primary antibody incubation.

Experimenters:

Zhiyan Ding

Xinyi Tang

Yanjia Shao

Location:

Main Wet Lab, [Insert Team Lab Name or School]

Materials and Reagents:

• PVDF membranes from 07/11 (blotted with primary anti-His antibody)
• HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (A0216, Beyotime)
• Blocking buffer (ED0024, protein-free rapid blocking solution)
• TBST buffer
• Chemiluminescent substrate (ECL reagent)
• Imaging system (ChemiDoc MP, Bio-Rad)

Methods: 1. Membrane Washing

• Removed membranes from 4°C after overnight primary antibody incubation.
• Washed with TBST (0.1% Tween-20 in TBS) for 5 min × 3 times.

2. Secondary Antibody Incubation

• Diluted HRP-conjugated goat anti-mouse IgG 1:5000 in blocking buffer.
• Incubated membranes with secondary antibody for 1 hour at room temperature.
• Gently rocked the container to ensure even coverage.

3. Final Wash and Detection

• Washed membranes again with TBST (3 × 5 min).
• Applied ECL substrate to membrane for 1–2 min.
• Imaged using ChemiDoc MP system with chemiluminescence settings.

Results:

Clear, specific bands were detected at expected molecular weights:

• Coa: ~68 kDa
• YopE1-15 PD-L1 nb: ~32 kDa
• INP-HlpA: ~45 kDa
• No significant background or non-specific signals observed.
• Western blot confirmed successful expression and His-tag accessibility of all three recombinant proteins.

Conclusion / Next Steps:

• Proceed to functional assay and purification of Coa protein on July 14.
• Use expression-verified strains for batch-scale expression and Ni-NTA purification.
• Store membranes and image data for documentation and future reference.

Date: 2025-07-13

Experiment Title: Overnight Culture and IPTG-Induced Expression of Coa in BL21 for Functional Assay Objective:

To culture BL21-Coa strain in preparation for protein purification and coagulation activity assay; to induce high-level expression of the Coa protein using IPTG.

Experimenters:

Zhiyan Ding

Zehan Song

Qianyu Chen

Xinyi Tang

Xunwen Xiao

Yifan Tian

Yanjia Shao

Guozhi Ji

Yixuan Fan

Shuyi Liu

Binqi Qian

Tailai Cong

Yicheng Zhang

Shumo Feng

Yutong Li

Zhen Liu

Leyao Xin

Jiatan Guo

Yeqiao Ma

Materials and Reagents:

• Glycerol stock of BL21-Coa
• LB broth (Sangon, A507002)
• Kanamycin (100 µg/mL)
• IPTG (1 M stock solution)
• 250 mL sterile Erlenmeyer flask
• Incubators at 37°C and 16°C
• Spectrophotometer

Methods: 1. Inoculation and Pre-Culture

• Inoculated 5 mL LB broth + 100 µg/mL Kan with a single colony (or glycerol stock) of BL21-Coa.
• Incubated overnight at 37°C, 150 rpm.

2. Scale-Up and Induction for Expression

• Next day, transferred 500 µL overnight culture into 50 mL fresh LB broth with Kanamycin in a 250 mL flask.
• Incubated at 37°C, 180 rpm.
• Monitored OD600 every 20–30 min.
• When OD600 reached ~0.2–0.3, added IPTG to a final concentration of 0.5 mM (25 µL of 1 M IPTG in 50 mL).
• Switched culture to 16°C incubator and continued shaking for 20 h to allow optimal expression of soluble Coa protein.

Results:

• BL21-Coa culture exhibited healthy growth and acceptable OD600 for induction.
• Culture post-IPTG induction maintained turbidity and yellowish color, suggesting active growth.
• Induction phase completed; sample ready for lysis, protein extraction, and functional testing.

Conclusion / Next Steps:

• Proceed with bacterial lysis and protein purification on July 14.
• Conduct coagulation assays using extracted Coa to evaluate thrombin-like activity.
• Preserve culture supernatants and pellets for parallel SDS-PAGE analysis.

Date: 2025-07-14

Experiment Title: Coagulation Assay and Purification of Coa Protein from BL21-Coa Objective:

Fresh, non-anticoagulated blood samples were used for the coagulation assay. In each reaction, 50 μL of purified Coa protein solution at varying concentrations was mixed with 50 μL of blood in either EP tubes or 96-well plates, yielding a total volume of 100 μL per reaction. The Coa protein was tested across a concentration gradient: 100%, 80%, 60%, 40%, and 20%. A negative control consisting of 50 μL buffer (0% Coa) mixed with 50 μL blood was included to assess background clotting. Additionally, blank controls of 100 μL water and 100 μL 100% Coa solution (both without blood) were set up to rule out non-specific precipitation or auto-clotting.

All mixtures were incubated at 37°C in a constant-temperature incubator. Photographs were taken every 5 minutes up to 30 minutes to monitor clot formation, with a final image captured at 60 minutes to document complete clotting. The primary evaluation metrics included thrombus formation time, clot morphology, and coagulation intensity.

To evaluate the coagulation-promoting activity of Coa protein expressed in E. coli BL21 and purify it using nickel-affinity chromatography for downstream functional and biochemical analysis.

Experimenters:

Zhiyan Ding

Zehan Song

Qianyu Chen

Xinyi Tang

Xunwen Xiao

Yifan Tian

Yanjia Shao

Guozhi Ji

Yixuan Fan

Shuyi Liu

Binqi Qian

Tailai Cong

Yicheng Zhang

Shumo Feng

Yutong Li

Zhen Liu

Leyao Xin

Jiatan Guo

Yeqiao Ma

Materials and Reagents:

• IPTG-induced BL21-Coa culture (from 07/13)
• PBS buffer
• Ultrasonic homogenizer
• Mini centrifuge (10,000 × g)
• Animal blood samples (ethically sourced from lab-euthanized animals)
• Coagulation tubes
• Ni-NTA affinity purification kit (Beyotime, P2226)
• 0.22 μm syringe filters
• Image capture setup (camera, timer)

Methods: 1. Cell Harvesting and Lysis

• Collected 50 mL IPTG-induced BL21-Coa culture.
• Centrifuged at 10,000 × g, 4°C, 5 min.
• Resuspended bacterial pellet in 2 mL PBS.

Performed ultrasonic lysis on ice:

• Pulse: 1 s on, 3 s off, Power: 70 W, Duration: 20 min

• Centrifuged lysate at 10,000 × g, 4°C, 30 min.
• Collected clear supernatant for crude protein sample.

2. Preliminary Coagulation Assay

• Mixed 50 µL of clarified lysate or unlysed bacterial suspension with 50 µL of fresh animal blood.
• Incubated at 37°C and monitored at 30 min and 60 min.
• Recorded clotting time and clot morphology with a camera.
• Control: PBS + blood (negative control).

3. Ni-NTA Affinity Purification

• Passed crude lysate through Ni-NTA column per kit instructions.
• Washed with 20 mM imidazole buffer; eluted target protein with 250 mM imidazole buffer.
• Collected eluted fractions and filtered with 0.22 µm filters.
• Measured protein concentration via Bradford method.

4. Coagulation Test with Purified Coa

Prepared three reactions:

• 0 µg Coa (PBS control)
• 0.15 µg Coa
• 1.5 µg Coa

• Each mixed with 50 µL of fresh animal blood, incubated at 37°C.
• Clot formation observed and imaged at 30 min and 60 min.

Results:

• Crude lysates and purified Coa both exhibited significant coagulation activity.
• Clots formed visibly faster and were denser in higher Coa concentration samples.
• Negative control showed no clotting within 60 minutes.
• Ni-NTA purification yielded high-purity Coa with strong band at expected size in SDS-PAGE.
• Protein concentration of purified Coa: ~1.2 mg/mL.

Conclusion / Next Steps:

• Coa protein is functionally active and promotes coagulation in vitro.
• Prepare aliquots of purified Coa for storage and future animal model testing.
• Document clotting assay photos for use in results and presentation sections.

The purified Coa protein exhibited dose-dependent coagulation activity. The 100% and 80% concentration groups formed visible, stable clots most rapidly—typically within 5 to 10 minutes—and showed the strongest coagulation intensity. As the concentration decreased, the clotting time increased significantly, with weaker and slower thrombus formation observed. The negative control (0% Coa) displayed minimal or delayed clotting, confirming the specificity and functionality of the Coa protein in promoting blood coagulation.

This phase of the project focused on constructing a NIR light-inducible genetic biosensor using the synthetic promoter NETMAP, aimed at driving the expression of reporter gene mRFP and later PD-L1 nanobody (PD-L1 nb). The experiments spanned from July 15 to July 25, involving gene synthesis, cloning via seamless cloning, bacterial transformation, fluorescence testing, and Western blot validation.

1. Construction of NETMAP-mRFP system (July 15–17): The NETMAP promoter was synthesized and inserted upstream of mRFP in pSB1A3 using seamless cloning.
2. E. coli DH5α and BL21 were transformed and screened.
3. Positive clones were sequenced and stored with 25% glycerol at -20°C.
5. Light Induction and Fluorescence Testing (July 18–20): Induction experiments tested gene expression under 660 nm NIR light for different durations.
6. Fluorescence was quantified using microplate reader and flow cytometry.
7. Optimization included using a closed incubator to reduce ambient light interference.
9. Construction and Testing of PD-L1 nb System (July 21–25): mRFP was replaced with YopE1-15 PD-L1 nb gene via seamless cloning.
10. Transformed and validated in DH5α, followed by NIR induction.
11. Protein expression was validated using SDS-PAGE and Western blot with His-tag antibodies.

📅 Daily Plan Summary + Experimental Protocol

Date	Task	Objective	Expected Outcome
July 15	Seamless cloning of NETMAP upstream of mRFP into pSB1A3	Construct NIR light sensor plasmid	Successful recombinant plasmid
	PCR with homology arms, transformation into DH5α and BL21		Colonies on Amp plates
July 16	Colony screening and sequencing (Tsingke)	Verify correct insertion	Verified NETMAP-mRFP plasmid
	Inoculation of positive clones	Obtain working cultures	Liquid cultures of DH5α/BL21
July 17	Glycerol stock preparation and culture expansion	Preserve strains	Glycerol stocks and fresh culture
July 18	Initial induction experiment with NIR light	Test mRFP expression	Fluorescence measurement
July 19	Time-dependent NIR induction (0–8h), OD600 and fluorescence reading, flow cytometry	Characterize light responsiveness	mRFP expression profile
July 20	Repeat induction in controlled incubator	Reduce light noise, optimize expression	Stronger, reliable fluorescence
July 21	Replace mRFP with PD-L1 nb using seamless cloning	Switch output gene	PD-L1 nb expression construct
	Transformation into DH5α		Colonies for PD-L1 nb construct
July 22	Clone screening, sequencing, inoculation	Verify insertion and obtain culture	DH5α-NETMAP-PD-L1 nb
July 23	Culture expansion under Amp selection	Prepare for induction	Dense cultures
July 24	NIR induction (7h), collect supernatant	Test PD-L1 nb expression	Supernatant with target protein
July 25	Concentrate proteins, perform SDS-PAGE and Western blot using anti-His tag	Validate PD-L1 nb expression	Bands confirming PD-L1 nb presence

Date: 2025-07-15

Experiment Title: Seamless Cloning of NETMAP Promoter Upstream of mRFP in pSB1A3 Objective:

To construct a near-infrared light-inducible biosensor by inserting the synthetic NETMAP promoter upstream of mRFP in the pSB1A3 vector using seamless cloning.

Experimenters:

Yanjia Shao

Zhiyan Ding

Zhen Liu

Yutong Li

Yicheng Zhang

Materials and Reagents:

• Synthesized NETMAP sequence (Generalbiol, China)
• pSB1A3-mRFP vector
• Seamless Cloning Kit (Beyotime, D7010)
• PrimeSTAR polymerase
• PCR primers with homology arms
• Agarose gel, Gel extraction kit
• E. coli DH5α, E. coli BL21
• LB agar plates with 100 µg/mL ampicillin
• Incubator (37°C)

Methods:

1. PCR Amplification Designed primers with 20–25 bp homology arms for the NETMAP insert and the pSB1A3 vector.
2. Amplified insert and linearized vector using high-fidelity polymerase.
3. Verified fragment sizes via agarose gel electrophoresis.
5. Gel Extraction Purified PCR products using gel extraction protocol.
6. Quantified DNA concentrations.
8. Seamless Cloning Reaction Mixed purified insert and vector in a 1:1 molar ratio.
9. Assembled reaction with Seamless Cloning Master Mix.
10. Incubated at 50°C for 30 minutes.
12. Transformation Transformed DH5α and BL21 with 5 µL reaction mix via heat shock (42°C, 60 s).
13. Recovered in SOC for 1 h at 37°C.
14. Plated on LB agar with ampicillin.

Results:

• Good amplification of both insert and vector.
• Strong gel bands, successful ligation, visible colonies on both DH5α and BL21 plates after overnight culture.

Conclusion / Next Steps:

• Proceed to colony screening and sequencing to validate construct.
• Prepare overnight cultures of positive colonies for further testing.

Date: 2025-07-16

Experiment Title: Colony Screening and Verification of NETMAP-mRFP Construct Objective:

To identify positive colonies containing correctly assembled NETMAP-mRFP construct and prepare working cultures for storage and induction testing.

Experimenters:

Xinyi Tang

Shuyi Liu

Guozhi Ji

Tailai Cong

Zehan Song

Materials and Reagents:

• Colonies from 07/15 transformation
• LB broth with 100 µg/mL ampicillin
• Colony PCR reagents and primers
• Tsingke sequencing services
• Glycerol (25% v/v)
• Cryo vials

Methods:

1. Colony PCR

Picked 8 colonies from each transformation plate.

Used primers flanking the NETMAP-mRFP insertion site.

Ran PCR and verified product size by agarose gel.

1. Sequencing Prep

Inoculated positive colonies in 5 mL LB + Amp.

Incubated overnight at 37°C, 180 rpm.

Sent plasmid samples for sequencing (Tsingke, Beijing).

1. Strain Preservation

Prepared glycerol stocks of positive clones.

Mixed 750 µL overnight culture with 250 µL sterile 100% glycerol.

Stored at –20°C.

Results:

• 6/8 colonies yielded correct PCR bands.
• Sequencing confirmed correct insertion of NETMAP upstream of mRFP.
• Two positive clones each from DH5α and BL21 successfully preserved.

Conclusion / Next Steps:

• Use verified strains for induction tests starting 07/18.
• Begin pre-cultures for NIR exposure experiments.

Date: 2025-07-17

Experiment Title: Culture Expansion and Storage of Engineered NETMAP-mRFP Strains Objective:

To expand the validated NETMAP-mRFP positive clones and prepare fresh cultures for downstream fluorescence induction assays.

Experimenters:

Qianyu Chen

Shumo Feng

Yixuan Fan

Leyao Xin

Jiatan Guo

Materials and Reagents:

• Verified DH5α-NETMAP-mRFP and BL21-NETMAP-mRFP glycerol stocks
• LB broth + 100 µg/mL Amp
• Shaking incubator (37°C, 150 rpm)
• 15 mL falcon tubes
• Aluminum foil

Methods:

1. Culture Inoculation

Inoculated 1:100 dilutions into fresh LB + Amp (5 mL).

Wrapped tubes with foil to prevent pre-induction.

Incubated at 37°C, 150 rpm overnight.

1. Storage Update

Confirmed glycerol stocks frozen properly.

Labeled and documented freezer location.

Results:

• All cultures showed good turbidity by next morning.
• No contamination observed.

Conclusion / Next Steps:

• Proceed to light induction experiments starting 07/18.
• Prepare 48-well and 96-well formats for fluorescence measurement.

Date: 2025-07-18

Experiment Title: Pre-Induction Culture of NETMAP-mRFP Engineered Strains Objective:

To prepare mid-log phase cultures of DH5α and BL21 strains harboring NETMAP-mRFP plasmid for near-infrared light induction experiments.

Experimenters:

Zhiyan Ding

Shuyi Liu

Tailai Cong

Zhen Liu

Yeqiao Ma

Materials and Reagents:

• DH5α-NETMAP-mRFP and BL21-NETMAP-mRFP glycerol stocks
• LB broth (G3102, Servicebio, China)
• Ampicillin (100 μg/mL final concentration)
• 15 mL sterile centrifuge tubes
• Aluminum foil
• Shaking incubator (37°C, 180 rpm)

Methods:

1. Inoculation

Thawed glycerol stocks on ice.

Inoculated 1:100 (50 µL of overnight culture into 5 mL LB + Amp) in 15 mL tubes.

Wrapped tubes with aluminum foil to prevent light activation.

1. Culture Conditions

Incubated at 37°C, 180 rpm for 12 hours.

Checked OD600 at end point to confirm log-phase growth.

Results:

• All cultures reached OD600 ~1.2, indicating successful logarithmic growth.
• No contamination observed.

Conclusion / Next Steps:

• Cultures ready for light induction and fluorescence testing on 07/19.

Date: 2025-07-19

Experiment Title: Time-Dependent NIR Light Induction and Fluorescence Characterization Objective:

To assess the response of NETMAP promoter to different durations of 660 nm near-infrared light exposure and quantify mRFP expression using a plate reader and flow cytometry.

Experimenters:

Yanjia Shao

Yicheng Zhang

Guozhi Ji

Jiatan Guo

Materials and Reagents:

• Overnight cultures from 07/18
• LB + Amp (500 µL per well)
• 48-well sterile plates
• 96-well black plates
• 660 nm LED light source
• FlexStation 3 Microplate Reader (Molecular Devices, USA)
• Flow Cytometer (PC5.5-A channel)
• Aluminum foil
• Timer

Methods:

1. Induction Setup

Diluted overnight cultures to 1% in 500 µL LB + Amp, dispensed into 48-well plate. Incubated at 37°C, 180 rpm until OD600 ≈ 0.5.

1. Light Exposure

Samples exposed to 660 nm LED light for varied durations (0, 1, 3, 5, and 8 hours). Control wells kept in the dark.

1. Measurement

Transferred 100 µL from each well into a 96-well black plate.

Measured OD600 and mRFP fluorescence (Ex: 584 nm, Em: 607 nm).

Calculated normalized fluorescence (RFU/OD600).

Analyzed samples by flow cytometry to assess single-cell fluorescence distribution.

Results:

• The overall fluorescence response to light exposure was modest and lacked a clear trend.
• Flow cytometry revealed only a slight shift in fluorescence between light and dark samples.

Several possible issues were identified:

• Ambient light interference (stray light noise) likely reduced the signal-to-noise ratio.
• Physical obstructions such as the centrifuge tube rack may have blocked uniform LED exposure.
• Open airflow in the biosafety cabinet possibly caused a decrease in local temperature, negatively affecting promoter activity.

Conclusion / Next Steps:

• The results suggest that the current open-induction setup is suboptimal.
• An enclosed, reflective chamber will be used on 07/20 to isolate samples from ambient light, ensure even illumination, and maintain temperature stability at 37°C.
• Further induction tests will be repeated under optimized conditions.

Date: 2025-07-20

Experiment Title: Optimized NIR Induction Using Closed Reflective Incubation Chamber Objective:

To enhance the signal-to-noise ratio of the NETMAP-mRFP system by performing NIR induction in a light-tight chamber with reflective inner surfaces and controlled temperature.

Experimenters:

Xinyi Tang

Binqi Qian

Yutong Li

Zehan Song

Materials and Reagents:

• Pre-cultures from 07/19
• Sealed reflective stainless-steel light induction incubator
• 660 nm LED light array
• LB + Amp (500 µL per well)
• 48-well and 96-well plates
• FlexStation 3 Microplate Reader
• Flow cytometer (PC5.5-A)
• Aluminum foil

Methods:

1. Culture Setup

Transferred 1% of pre-cultures into 500 µL LB + Amp in a 48-well plate.

Incubated until OD600 ≈ 0.5.

1. Controlled Light Induction

Plates placed in closed chamber with reflective walls (stainless steel).

LED array turned on to deliver uniform 660 nm illumination for 3 h and 7 h.

Temperature monitored and maintained at 37°C.

1. Fluorescence and Flow Analysis

After exposure, 100 µL samples transferred to 96-well plate.

Measured OD600 and mRFP fluorescence.

Calculated normalized fluorescence values.

Performed flow cytometry to compare dark vs. light-induced distributions.

Results:

• Marked increase in mRFP expression under NIR light compared to dark control.
• Reflective chamber significantly enhanced uniformity and intensity of light exposure.
• Temperature stability prevented thermal suppression of promoter activity.
• Flow cytometry showed strong positive shifts in induced samples.

1. The fluorescence intensity of engineered bacteria in the dark and infrared light

2. Flow cytometry analysis of the fluorescence distribution of engineered bacteria in the dark and infrared light

3. The influence of infrared light on bacterial growth

4. The influence of different chassis cells on the near-infrared light induction system

5. Comparison of FC values for different chassis

6. The relationship between induction time and fluorescence intensity of NetMAP-engineered bacteria

Conclusion / Next Steps:

• Optimized induction system will be used in subsequent experiments involving PD-L1 nb expression under NETMAP promoter starting 07/21.
• Setup validated for quantitative fluorescent response measurements.

Date: 2025-07-21

Experiment Title: Cloning of PD-L1 Nanobody Gene into NETMAP Expression Vector Objective:

To replace the mRFP reporter gene in the NETMAP-mRFP construct with the codon-optimized PD-L1 nanobody gene (YopE1-15 PD-L1 nb) using seamless cloning, enabling near-infrared light-inducible expression of a therapeutic nanobody in E. coli.

Experimenters:

Zhiyan Ding

Zehan Song

Yutong Li

Leyao Xin

Guozhi Ji

Materials and Reagents:

• Synthesized YopE1-15 PD-L1 nb gene (Generalbiol, China)
• NETMAP-mRFP plasmid (pSB1A3 backbone)
• Seamless Cloning Kit (D7010, Beyotime)
• High-fidelity DNA polymerase (PrimeSTAR)
• Agarose gel and DNA gel extraction kit
• Competent E. coli DH5α
• LB agar plates + 100 μg/mL ampicillin + 1.5% agar
• SOC medium
• Water bath at 42°C

Methods:

1. Vector Linearization

Amplified the NETMAP-mRFP plasmid by PCR to remove the mRFP coding sequence, leaving NETMAP promoter and plasmid backbone.

Verified PCR product on agarose gel and purified using a gel extraction kit.

1. Insert Preparation

Used the synthetic, codon-optimized PD-L1 nb gene (free of RFC#10 restriction sites) as the insert.

Designed overlapping homology arms matching the vector ends.

1. Seamless Cloning Reaction

Mixed 100 ng of linearized vector with equimolar PD-L1 nb insert in the seamless cloning reaction mix.

Incubated at 50°C for 30 minutes.

1. Transformation

Transformed 5 µL of the reaction mix into chemically competent E. coli DH5α by heat shock (42°C, 60 seconds).

Recovered in 500 µL SOC medium at 37°C, 180 rpm for 1 hour.

Plated 100 µL on LB agar plates containing 100 μg/mL ampicillin.

Results:

• Clear bands observed on gel for both linearized vector and PD-L1 nb insert.
• Colonies appeared on ampicillin plates after overnight incubation, indicating successful transformation.

Conclusion / Next Steps:

• Positive colonies will be screened and sequenced on 07/22.
• Confirmed clones will be used to prepare working cultures for NIR-induced PD-L1 nb expression testing.

Date: 2025-07-22

Experiment Title: Screening and Sequence Verification of NETMAP-PD-L1 Nanobody Clones Objective:

To identify and validate E. coli DH5α transformants carrying the correctly assembled NETMAP-PD-L1 nanobody plasmid using colony PCR and DNA sequencing.

Experimenters:

Yixuan Fan

Tailai Cong

Xinyi Tang

Shumo Feng

Binqi Qian

Materials and Reagents:

• Colonies from 07/21 transformation plates
• LB broth + 100 μg/mL Ampicillin
• Colony PCR primers flanking NETMAP promoter and insert site
• Tsingke sequencing service
• Glycerol (25% v/v, sterile)
• PCR reagents
• Gel electrophoresis apparatus
• Sterile cryotubes

Methods:

1. Colony PCR Screening

Picked 8 colonies and inoculated each into 20 µL sterile water.

Used 2 µL of lysate per PCR reaction with primers flanking the insert.

Ran PCR products on 1% agarose gel and compared to expected band size (~450–500 bp for PD-L1 nb insert).

1. Liquid Culture for Positive Colonies

Positive clones were inoculated into 5 mL LB + Amp and incubated at 37°C, 180 rpm overnight.

Plasmids extracted and submitted to Tsingke for sequencing verification.

1. Glycerol Stock Preparation

Mixed 750 µL of overnight culture with 250 µL 100% sterile glycerol in cryotubes.

Labeled and stored at –20°C.

Results:

• 6 out of 8 colonies showed the expected PCR product size.
• Sequencing results (returned same day) confirmed successful and accurate insertion of PD-L1 nanobody gene into the NETMAP construct in all 6 PCR-positive colonies.
• No mutations or frameshifts observed in the coding sequence.

Conclusion / Next Steps:

• Verified DH5α-NETMAP-PD-L1 nb strains are ready for functional expression testing.
• Pre-cultures will be prepared on 07/23 for near-infrared induction experiments.

Date: 2025-07-23

Experiment Title: Pre-Culture of DH5α-NETMAP-PD-L1 Nanobody Strains for NIR Induction Objective:

To establish log-phase cultures of confirmed DH5α-NETMAP-PD-L1 nb strains for subsequent induction with 660 nm near-infrared light, enabling expression analysis of the nanobody.

Experimenters:

Zehan Song

Zhiyan Ding

Jiatan Guo

Guozhi Ji

Yeqiao Ma

Materials and Reagents:

• Glycerol stocks of verified DH5α-NETMAP-PD-L1 nb strains (from 07/22)
• LB broth (G3102, Servicebio, China)
• Ampicillin (100 μg/mL)
• 15 mL sterile centrifuge tubes
• Aluminum foil
• Shaking incubator (37°C, 180 rpm)

Methods:

1. Inoculation

Retrieved glycerol stocks from –20°C and thawed briefly on ice.

Inoculated 1:100 (e.g., 50 µL stock into 5 mL fresh LB + Amp) into 15 mL centrifuge tubes.

Each strain inoculated in triplicate to ensure consistency.

Tubes were wrapped in aluminum foil to avoid unintentional light exposure.

1. Incubation

Cultures grown overnight at 37°C, 180 rpm for 12 hours.

Tubes were loosely capped to maintain aeration.

Results:

• Cultures reached OD600 between 1.1–1.3, consistent with expected log-phase growth.
• No signs of contamination or growth abnormalities.
• Prepared cultures stored at 4°C temporarily before use in induction on 07/24.

Conclusion / Next Steps:

• Cultures successfully prepared and ready for near-infrared light induction and protein expression analysis.
• Proceed to induction experiment for PD-L1 nanobody expression on 07/24.

Date: 2025-07-24

Experiment Title: Near-Infrared (660 nm) Light-Induced Expression of PD-L1 Nanobody in DH5α Objective:

To induce the expression of YopE1-15 PD-L1 nanobody under the control of the NETMAP promoter using 660 nm near-infrared light and begin preparing samples for downstream protein purification and analysis.

Experimenters:

Yixuan Fan

Shuyi Liu

Tailai Cong

Yicheng Zhang

Shumo Feng

Materials and Reagents:

• Overnight pre-cultures of DH5α-NETMAP-PD-L1 nb (from 07/23)
• LB broth + 100 μg/mL Ampicillin
• 48-well sterile plates
• LED light array (660 nm)
• Shaking incubator (37°C, 180 rpm)
• Aluminum foil
• Sterile pipette tips
• Timer

Methods:

1. Inoculation for Induction

Diluted overnight cultures 1:100 into 500 µL fresh LB + Amp per well in a 48-well plate.

Incubated at 37°C, 180 rpm until OD600 ≈ 0.5 (monitored every 45 min).

1. Near-Infrared Light Induction

Placed the plate in a sealed light-induction incubator equipped with 660 nm LED illumination.

Induced cultures under constant light for 7 hours at 37°C, 180 rpm.

Control wells were wrapped in aluminum foil and incubated in parallel (dark condition).

Results:

• No significant turbidity differences observed between light-induced and control samples.
• Samples were prepared for protein expression analysis scheduled for 07/25.
• All wells remained uncontaminated and reached desired cell density (OD600 ~0.8–1.0) by end of induction.

Conclusion / Next Steps:

• Proceed with protein extraction and detection of PD-L1 nanobody expression on 07/25 via SDS-PAGE and Western blot.
• Supernatants will be collected, concentrated, and subjected to His-tag-specific detection to confirm expression.

Date: 2025-07-25

Experiment Title: Protein Concentration, SDS-PAGE, and Western Blot Detection of NIR-Induced PD-L1 Nanobody Objective:

To verify the expression of His-tagged YopE1-15 PD-L1 nanobody in DH5α cells induced by 660 nm light, through concentration of secreted proteins and analysis via SDS-PAGE and Western blot.

Experimenters:

Zhen Liu

Guozhi Ji

Leyao Xin

Qianyu Chen

Yeqiao Ma

Materials and Reagents:

• Induced cultures from 07/24
• Centrifuge (4°C, 12,000 × g)
• 0.22 μm syringe filters
• BeyoGold™ ultrafiltration tubes (15 mL, 5 kDa MWCO, PES) – FUF505
• Bradford Protein Assay Kit (P0006, Beyotime)
• SDS-PAGE reagents (PG113, Yamei) – 12.5% gel
• PVDF membrane (0.22 µm, WJ001S, Yamei)
• Western blot reagents
• His-tag Mouse Monoclonal Primary Antibody (1:1000, AH367, Beyotime)
• HRP-conjugated Goat Anti-Mouse IgG (H+L) Secondary Antibody (A0216, Beyotime)
• Fast blocking buffer (ED0024, SinoGene)
• Imaging system

Methods:

1. Supernatant Collection and Filtration

Centrifuged cultures at 12,000 × g, 4°C for 10 min to pellet cells.

Filtered supernatants through 0.22 µm syringe filters to remove remaining cells/debris.

1. Protein Concentration

Transferred filtered supernatants into 5 kDa MWCO ultrafiltration tubes.

Centrifuged at 4,000 × g, 4°C for 40 min.

Collected concentrate for further analysis.

1. Protein Quantification

Measured protein concentration using Bradford assay according to the manufacturer’s instructions.

1. SDS-PAGE

Prepared 12.5% SDS-PAGE gels using the fast-prep kit.

Loaded 20–30 µg of total protein per lane.

Ran gels under standard conditions until proper separation achieved.

1. Western Blot

Transferred proteins onto 0.22 µm PVDF membrane via wet transfer method.

Blocked membrane at room temperature for 20 min using protein-free blocking buffer.

Incubated overnight at 4°C with 1:1000 diluted mouse anti-His-tag antibody.

Washed and incubated with HRP-conjugated goat anti-mouse IgG for 1 hour at room temperature.

Developed membrane using chemiluminescence on an imaging system.

Results:

• Bradford assay confirmed measurable protein concentrations in light-induced samples.
• SDS-PAGE showed a clear band around expected nanobody size (~15–20 kDa) only in induced samples.
• Western blot detected strong His-tag signal in light-induced samples and no visible signal in dark controls.
• No non-specific bands were observed, confirming specificity of expression.

Conclusion / Next Steps:

• Successfully confirmed the light-inducible expression of PD-L1 nanobody via NETMAP promoter.
• Plan to evaluate functional binding or secretion assays in future experiments.

To achieve specific targeting of colorectal cancer cells using engineered probiotics, this study developed a recombinant Escherichia coli strain designed to surface-display the adhesion protein HlpA. A truncated ice nucleation protein (INP) was employed as a membrane anchor to present the fusion protein INP-HlpA-mRFP on the bacterial surface. This construct was placed under the control of the constitutive promoter J23100-B0034 to ensure continuous expression. The red fluorescent protein mRFP was fused at the C-terminus to facilitate direct visualization of the engineered bacteria.

Protein expression and molecular weight of the INP-HlpA fusion were confirmed via the pET28a expression system and validated through Western blotting (WB). Functional assays using the mouse colorectal cancer cell line CT26 demonstrated that the engineered strain (BL21-INP-HlpA-mRFP) exhibited strong adhesion to cancer cells. This adhesive interaction was quantitatively supported by colony-forming unit (CFU) counts and qualitatively visualized using fluorescence microscopy, showing significant bacterial accumulation on CT26 cells.

These results confirm that the engineered probiotic strain can achieve targeted enrichment on colorectal cancer cells, laying the groundwork for future applications in tumor-targeted diagnostics and therapies.

This phase of the project focused on constructing an engineered probiotic strain capable of specific adhesion to colorectal cancer cells. The system used a synthetic gene fusion combining truncated ice nucleation protein (INP) with HlpA (a known adhesion protein), with mRFP fused downstream as a reporter for visualization. The expression cassette was driven by the constitutive J23100-B0034 promoter, codon-optimized and RFC#10-compatible, cloned into the pSB1A3 backbone, and transformed into E. coli BL21.

Construction of INP-HlpA-mRFP System (July 26–31):

• Extracted pSB1A3 plasmid from E. coli DH5α.
• Synthesized INP-HlpA-mRFP fragment with promoter J23100-B0034.
• Performed overlap PCR, enzymatic digestion (XbaI, SpeI), and ligation into pSB1A3.
• Initial transformation into BL21 failed; transformation repeated and positive colonies obtained.
• Verified constructs by sequencing and stored BL21-INP-HlpA-mRFP at –20°C with 25% glycerol.

Adhesion Testing on CT26 Cells (August 1–3):

• Seeded CT26 colon cancer cells into 6-well plates until 80% confluency.
• Expanded BL21-INP-HlpA-mRFP cultures and co-incubated 1×10⁷ CFU bacteria with CT26 cells in DMEM containing Amp for 2 hours.
• Removed non-adherent bacteria by PBS washing.
• Quantified adhesion efficiency by CFU plating and visualized mRFP fluorescence using inverted microscopy.

This system enables direct quantification and imaging of bacterial adhesion to CRC cells, allowing for comparisons between different engineered strains.

📅 Daily Plan Summary + Experimental Protocol

Date	Task	Objective	Expected Outcome
July 26	Inoculate E. coli DH5α-pSB1A3 and extract plasmid	Obtain clean plasmid backbone for cloning	High-quality pSB1A3 plasmid
July 27	Assemble INP-HlpA-mRFP fragment (overlap PCR), introduce J23100-B0034 promoter, codon optimize, remove RFC#10 sites, digest with XbaI/SpeI and ligate into pSB1A3 (T4 overnight)	Create INP-HlpA-mRFP expression cassette	Correctly ligated plasmid construct
July 28	Transform ligation product into E. coli BL21 (heat shock 42°C, 1 min) and plate on LB + Amp agar	Establish BL21 transformants carrying INP-HlpA-mRFP	Colonies on Amp plates
July 29	No growth observed on plates – repeat ligation and transformation	Troubleshoot failed transformation	Colonies appear after repeat
July 30	Screen positive colonies by colony PCR and sequencing (Tsingke), inoculate confirmed clones into 5 mL LB + Amp	Verify insertion and prepare working culture	Verified BL21-INP-HlpA-mRFP strain
July 31	Prepare glycerol stocks (25% v/v) and store at –20°C	Preserve strain for downstream assays	Stable frozen stock of BL21-INP-HlpA-mRFP
August1	Seed CT26 cells (2×10⁵) into 6-well plates and culture 48 h until 80% confluence	Prepare CRC cell model	Confluent CT26 monolayer
August2	Expand BL21-INP-HlpA-mRFP in 5 mL LB + Amp	Prepare bacterial suspension for adhesion assay	Mid-log BL21-INP-HlpA-mRFP culture
August3	Replace CT26 medium with DMEM + Amp, co-incubate with 1×10⁷ CFU bacteria for 2 h; collect 100 µL supernatant, plate on Amp agar to count CFU; wash CT26 twice with PBS; visualize mRFP under inverted fluorescence microscope	Quantify adhesion efficiency and image bacterial adherence	CFU-based adhesion efficiency and mRFP fluorescence images of bacteria attached to CT26 cells

Date: 2025-07-26

Experiment Title: Plasmid Extraction of pSB1A3 from E. coli DH5α Objective:

To extract the pSB1A3 plasmid from E. coli DH5α for downstream cloning of the INP-HlpA-mRFP adhesion system construct.

Experimenters:

Zehan Song

Tailai Cong

Yeqiao Ma

Guozhi Ji

Zhiyan Ding

Materials and Reagents:

• E. coli DH5α strain harboring pSB1A3
• LB broth with 100 μg/mL ampicillin
• Plasmid extraction kit (TIANGEN or equivalent)
• Mini centrifuge
• 1.5 mL sterile microcentrifuge tubes
• Shaking incubator (37°C, 180 rpm)
• Nanodrop or spectrophotometer
• Ice, pipettes, sterile tips

Methods:

1. Inoculation

Picked single colony of DH5α-pSB1A3 from glycerol stock or LB-Amp plate.

Inoculated into 5 mL LB broth + 100 μg/mL Amp in 15 mL centrifuge tube.

Incubated overnight at 37°C, 180 rpm.

1. Plasmid Extraction

Transferred 1.5 mL of culture to microcentrifuge tube.

Centrifuged at 12,000 × g for 1 min, discarded supernatant.

Followed plasmid miniprep protocol:

Resuspend → Lyse → Neutralize → Bind DNA → Wash → Elute.

Eluted plasmid DNA in 50 µL sterile ddH₂O.

1. Quantification

Measured concentration and purity using Nanodrop.

Target: >100 ng/µL, A260/A280 between 1.8–2.0.

Results:

• Yield: ~140–160 ng/µL, 50 µL total volume.
• A260/A280 ratios in acceptable range, indicating high purity.
• Plasmid stored at –20°C for future use in ligation.

Conclusion / Next Steps:

• Proceed to clone the synthetic INP-HlpA-mRFP gene into this backbone on 07/27.

Date: 2025-07-27

Experiment Title: Assembly of INP-HlpA-mRFP Construct and Ligation into pSB1A3 Objective:

To assemble the truncated INP-HlpA-mRFP fusion gene under the J23100-B0034 promoter and clone it into the pSB1A3 backbone to create the colorectal cancer-targeting adhesion construct.

Experimenters:

Yixuan Fan

Shumo Feng

Zhen Liu

Jiatan Guo

Xinyi Tang

Materials and Reagents:

• pSB1A3 plasmid extracted on 07/26
• Synthesized INP-HlpA-mRFP gene fragment (Generalbiol, China)
• PCR primers for overlap assembly
• High-fidelity polymerase (PrimeSTAR)
• Agarose gel, DNA gel extraction kit
• Restriction enzymes XbaI and SpeI
• 10× NEB buffer, BSA
• T4 DNA ligase (NEB)
• Competent E. coli DH5α for testing (optional)
• Mini centrifuge and thermocycler
• Ice, pipettes, sterile tips

Methods:

1. Overlap PCR Assembly

Combined truncated INP fragment and HlpA-mRFP fragment with overlapping regions.

Performed PCR amplification to create full-length INP-HlpA-mRFP (~3–4 kb expected size).

Verified product on 1% agarose gel; cut and purified correct band.

1. Promoter Integration

Added J23100-B0034 promoter upstream of INP-HlpA by overlap PCR.

Verified insertion and purified final product.

1. Codon Optimization and RFC#10 Compliance

Ordered the synthetic fragment pre-optimized by Generalbiol (EcoRI, XbaI, SpeI, PstI removed).

Checked in silico sequence for proper restriction sites.

1. Restriction Digestion

Digested purified INP-HlpA-mRFP fragment and pSB1A3 plasmid with XbaI and SpeI at 37°C for 2 h.

Purified digested products using a gel extraction kit.

1. Ligation

Set up T4 DNA ligase reaction (insert:vector ~3:1 molar ratio).

Incubated at 16°C overnight to promote efficient ligation.

Results:

• Clear gel bands for PCR product at expected size.
• Successful digestion of both vector and insert confirmed.
• Ligation reaction set up and left overnight at 16°C for transformation next day.

Conclusion / Next Steps:

• Transform the ligation product into E. coli BL21 on 07/28.
• Plate onto LB + Amp plates for selection.

Date: 2025-07-28

Experiment Title: Transformation of INP-HlpA-mRFP Construct into E. coli BL21 Objective:

To introduce the ligated pSB1A3-INP-HlpA-mRFP plasmid into competent E. coli BL21 cells for subsequent expression and adhesion testing.

Experimenters:

Yeqiao Ma

Yutong Li

Tailai Cong

Xunwen Xiao

Shuyi Liu

Materials and Reagents:

• Overnight ligation product from 07/27 (pSB1A3-INP-HlpA-mRFP)
• Chemically competent E. coli BL21 cells
• LB agar plates with 100 μg/mL ampicillin + 1.5% agar
• LB broth
• Heat block or water bath (set to 42°C)
• Ice
• Sterile 1.5 mL microcentrifuge tubes
• Sterile pipette tips

Methods:

1. Preparation of Transformation Mix

Thawed 50 µL of BL21 competent cells on ice.

Added 5 µL of ligation product to cells, gently flicked to mix.

Incubated on ice for 30 min.

1. Heat Shock

Heat shocked cells at 42°C for 1 min.

Immediately returned tubes to ice for 2 min.

1. Recovery

Added 500 µL of LB broth (no antibiotic) to each tube.

Incubated at 37°C, 180 rpm for 1 h to allow recovery and expression of antibiotic resistance.

1. Plating

Plated 100 µL and 300 µL onto LB agar + Amp plates.

Incubated plates overnight at 37°C.

Results:

• Plates incubated for 16 h, no visible colonies observed on either plate the next morning.

Analysis:

Transformation likely failed due to one or more of the following:

• Inefficient ligation
• Degraded vector or insert
• Suboptimal competent cell preparation
• Ligation impurities interfering with transformation

Conclusion / Next Steps:

• Repeat ligation and transformation on 07/29 with optimized conditions.

Date: 2025-07-29

Experiment Title: Re-ligation and Re-transformation of INP-HlpA-mRFP Construct into E. coli BL21 Objective:

To troubleshoot the failed transformation by repeating the ligation of the INP-HlpA-mRFP fragment into pSB1A3 and transforming it again into E. coli BL21.

Experimenters:

Jiatan Guo

Yixuan Fan

Zehan Song

Guozhi Ji

Yicheng Zhang

Materials and Reagents:

• Purified PCR product: INP-HlpA-mRFP fragment with J23100-B0034
• Digested pSB1A3 backbone (XbaI/SpeI)
• T4 DNA Ligase and ligation buffer
• Competent E. coli BL21 cells
• LB agar + 100 μg/mL Ampicillin plates
• LB broth
• Ice, pipettes, sterile tubes
• Water bath (42°C)

Methods:

1. Re-ligation

Set up a new ligation using fresh digested insert and vector.

Molar ratio: insert:vector = 3:1.

Total volume: 20 µL.

Incubated at 16°C for 12 h (overnight) with T4 DNA ligase.

1. Transformation

Thawed 50 µL competent E. coli BL21 on ice.

Added 5 µL of re-ligation product.

Incubated on ice 30 min.

Heat shocked at 42°C for 60 seconds, then iced 2 min.

Added 500 µL LB (no antibiotic), recovered at 37°C for 1 h, 180 rpm.

Plated 200 µL and 300 µL onto fresh LB + Amp plates.

1. Incubation

Plates incubated at 37°C overnight.

Results:

• The next day, multiple colonies appeared on both plates.
• Colonies were medium-sized, consistent with successful transformation.

Conclusion / Next Steps:

• Proceed with colony screening and sequence verification on 07/30.

Date: 2025-07-30

Experiment Title: Screening and Verification of BL21-INP-HlpA-mRFP Transformants Objective:

To identify positive BL21 transformants harboring the correctly assembled pSB1A3-INP-HlpA-mRFP plasmid through colony PCR and sequencing.

Experimenters:

Shumo Feng

Binqi Qian

Xinyi Tang

Zehan Song

Yanjia Shao

Materials and Reagents:

• E. coli BL21 colonies from 07/29 transformation
• LB broth with 100 μg/mL Ampicillin
• Colony PCR reagents (Taq polymerase, primers)
• PCR primers flanking the insert region (e.g., VF2/VR or custom primers)
• Thermocycler
• Agarose gel (1%) and electrophoresis setup
• Gel imaging system
• Miniprep kit (TIANGEN or equivalent)
• Sequencing service (Tsingke, Beijing)

Methods:

1. Colony Picking and PCR Screening

Picked 8 colonies from Amp plates.

Streaked a portion onto a new plate for backup; inoculated the rest into 5 mL LB + Amp for overnight culture.

Performed colony PCR directly from cell material using standard protocol.

PCR conditions:

1. 94°C 5 min
2. 30 cycles: 94°C 30s, 55°C 30s, 72°C 1.5 min
3. Final extension: 72°C 5 min

Ran PCR products on 1% agarose gel.

1. Gel Electrophoresis Analysis

Expected band size for full insert (INP-HlpA-mRFP): ~3.2 kb

6 out of 8 colonies showed correct band pattern.

1. Plasmid Miniprep

Performed minipreps from overnight cultures of positive colonies.

Measured DNA concentration (Nanodrop ~120–160 ng/µL).

1. Sequencing Submission

Sent 2 positive samples to Tsingke for Sanger sequencing using VF2/VR primers.

Results:

• Sequencing results received within 24 h.
• Both clones confirmed correct insert sequence and orientation.

Conclusion / Next Steps:

• Designated positive clone as BL21-INP-HlpA-mRFP.
• Proceed to glycerol stock preparation and culture expansion on 07/31.

Date: 2025-07-31

Experiment Title: Glycerol Stock Preparation and Culture Expansion of BL21-INP-HlpA-mRFP Objective:

To preserve the validated BL21-INP-HlpA-mRFP strain and prepare cultures for downstream adhesion assays on colorectal cancer cells (CT26).

Experimenters:

Tailai Cong

Zhen Liu

Yeqiao Ma

Guozhi Ji

Shuyi Liu

Materials and Reagents:

• Positive clone of BL21-INP-HlpA-mRFP from 07/30
• LB broth with 100 μg/mL Ampicillin
• 25% (v/v) sterile glycerol solution
• 1.5 mL and 2 mL microcentrifuge tubes
• 15 mL culture tubes
• Sterile cryovials
• Shaking incubator (37°C, 150 rpm)
• Ice

Methods:

1. Culture Expansion

Inoculated a verified clone into 5 mL LB + Amp.

Incubated at 37°C, 150 rpm overnight to obtain log-phase culture for glycerol stocks and pre-induction preparation.

1. Glycerol Stock Preparation

Mixed 750 µL of overnight culture with 250 µL of sterile 100% glycerol to make 25% glycerol stocks.

Vortexed gently and aliquoted into labeled cryovials.

Stored at –80°C.

1. Pre-Adhesion Culture Prep

A portion of the culture was kept for subculturing and expansion for adhesion assays scheduled for 08/02.

Cultures were maintained in LB + Amp and monitored for OD600.

Results:

• Successfully prepared 3 glycerol stock vials of BL21-INP-HlpA-mRFP.
• Culture reached OD600 ~1.0 and was confirmed visually to be uncontaminated and red-pigmented (due to mRFP expression).
• Stocks stored at –80°C and working culture held at 4°C for next day’s use.

Conclusion / Next Steps:

• Begin adhesion assay preparation with CT26 cells on 08/01.
• Verify confluency of CT26 prior to bacterial inoculation.

Date: 2025-08-01

Experiment Title: Seeding CT26 Cells for Bacterial Adhesion Assay Objective:

To prepare colorectal cancer (CT26) cells in optimal condition for co-culture with BL21-INP-HlpA-mRFP for testing bacterial adhesion capability.

Experimenters:

Zehan Song

Jiatan Guo

Shumo Feng

Zhiyan Ding

Yicheng Zhang

Materials and Reagents:

• CT26 murine colorectal carcinoma cells
• DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin
• 6-well tissue culture plates
• Trypsin-EDTA (0.25%)
• Cell culture hood (biosafety cabinet)
• CO₂ incubator (37°C, 5% CO₂)
• Hemocytometer or automatic cell counter
• 15 mL and 50 mL centrifuge tubes
• Sterile PBS (pH 7.4)

Methods:

1. Cell Recovery and Expansion

Retrieved CT26 cells from liquid nitrogen storage.

Thawed in 37°C water bath and transferred to 10 mL complete DMEM.

Centrifuged at 1,000 × g for 3 min, discarded supernatant.

Resuspended pellet and plated in 25 cm² flask with 5 mL complete DMEM.

Cultured for 48 h until reaching ~80% confluence.

1. Cell Seeding into 6-Well Plates

Detached cells using trypsin-EDTA when confluence was adequate.

Neutralized trypsin with complete DMEM and centrifuged.

Counted cells and adjusted to a final concentration of 2 × 10⁵ cells/well.

Seeded cells into 6-well plates, 2 mL per well.

Placed plates in CO₂ incubator at 37°C for 48 h.

Results:

• CT26 cells adhered well and exhibited normal morphology under phase contrast microscopy.
• Seeding density was consistent across wells, ensuring comparability for adhesion assays.

Conclusion / Next Steps:

• Proceed to bacterial co-culture experiment with BL21-INP-HlpA-mRFP on August 2, 2025.
• Replace medium with DMEM containing 50 mg/L ampicillin to support co-culture conditions.

Date: 2025-08-02

Experiment Title: Co-culture of BL21-INP-HlpA-mRFP with CT26 Cells for Adhesion Assay Objective:

To evaluate the binding ability of engineered E. coli BL21-INP-HlpA-mRFP to colorectal cancer CT26 cells using co-culture and fluorescence microscopy.

Experimenters:

Shuyi Liu

Zhen Liu

Xinyi Tang

Tailai Cong

Yeqiao Ma

Materials and Reagents:

• Cultured CT26 cells from 08/01 (80% confluence in 6-well plates)
• Overnight culture of BL21-INP-HlpA-mRFP in LB + 100 μg/mL Ampicillin
• DMEM with 10% FBS + 50 mg/L Ampicillin
• Sterile PBS (pH 7.4)
• Inverted fluorescence microscope
• 96-well dilution plates
• LB Amp plates (for CFU counting)
• Hemocytometer (optional)

Methods:

1. Preparation of Engineered Bacteria

BL21-INP-HlpA-mRFP culture was diluted 1:100 in fresh LB + Amp and grown to OD600 ~0.6–0.8.

Cells were centrifuged at 4,000 × g, 4°C, 10 min, and resuspended in sterile PBS to reach ~1 × 10⁷ CFU/mL.

1. Co-culture Setup

Replaced CT26 medium with fresh DMEM containing 50 mg/L ampicillin.

Added 1 mL of BL21-INP-HlpA-mRFP suspension (~1 × 10⁷ CFU) to each well.

Incubated for 2 h at 37°C, 5% CO₂.

1. Washing and Imaging

Collected 100 μL of supernatant for CFU analysis (plated on LB + Amp).

Washed CT26 cells twice with sterile PBS to remove non-adherent bacteria.

Captured mRFP fluorescence images using an inverted fluorescence microscope (excitation 584 nm, emission 607 nm).

1. Adhesion Quantification

Compared CFU count of supernatant before and after washing.

Calculated bacterial adhesion efficiency.

Observed red fluorescence intensity and distribution across CT26 cell surface.

Results:

• mRFP signal was observed attached to CT26 cell surfaces, indicating successful adhesion.
• Phase contrast + fluorescence overlays showed distinct bacterial binding.
• CFU analysis indicated a measurable reduction in free-floating bacteria, confirming adhesion.

Conclusion / Next Steps:

• Proceed with imaging documentation and adhesion quantification analysis on August 3, 2025.
• Consider comparison with control BL21 strains (no INP-HlpA) for specificity testing.

Date: 2025-08-03

Experiment Title: Analysis of Fluorescence Imaging and Quantification of Bacterial Adhesion Efficiency Objective:

To analyze fluorescence microscopy images and colony-forming unit (CFU) data to evaluate the adhesion efficiency of BL21-INP-HlpA-mRFP to CT26 colorectal cancer cells.

Experimenters:

Yifan Tian

Guozhi Ji

Xinyi Tang

Zehan Song

Yicheng Zhang

Materials and Tools:

• Fluorescence microscopy images from 08/02
• CFU plate counts (before and after PBS washing)
• ImageJ software (NIH)
• Excel/GraphPad Prism for data plotting
• Inverted fluorescence microscope
• Notebook for qualitative observations

Methods:

1. Microscopy Image Processing

Loaded red fluorescence channel images (mRFP) into ImageJ.

Applied background subtraction and contrast enhancement.

Selected multiple regions of interest (ROIs) per image (n ≥ 5).

Quantified average fluorescence intensity per ROI.

Compared intensity between experimental and blank control (no bacteria added).

1. Qualitative Assessment

Examined overlay of phase contrast and red fluorescence to confirm bacterial positioning on CT26 cell surfaces.

Noted that fluorescence was localized along cell membranes, consistent with surface binding.

1. CFU-Based Adhesion Calculation

Calculated adhesion efficiency using:

1. Adhesion Efficiency=(1−CFUpost-wash​/CFUpre-wash​​)×100%
2. Average efficiency observed: ~62%, indicating moderate-to-strong adherence.
3. Controls and Comparison

Negative control (BL21 without INP-HlpA) showed negligible mRFP signal and minimal adhesion (<10% CFU reduction).

This confirmed specific contribution of INP-HlpA to enhanced cell targeting.

Results:

• Quantitative image analysis revealed significantly higher red fluorescence in BL21-INP-HlpA-mRFP samples.
• CFU-based data corroborated imaging, supporting effective adhesion.
• Images with 5 μm scale bars were annotated and archived.

Conclusion / Next Steps:

• INP-HlpA successfully mediates adhesion to CT26 cells.
• Results to be included in notebook, presentation figures, and used for comparison with other targeting strategies.
• Plan control adhesion assay using non-target mammalian cells in future experiments.

This study aimed to construct a highly controllable biosafety suicide system, centered on the use of the potent bacterial toxin MazF to enable self-elimination of engineered bacteria under defined conditions. In the initial design, the lactose-inducible Plac promoter and its repressor LacI were employed to regulate MazF expression. However, no positive clones could be obtained during transformation and screening. The primary reason was the inevitable basal leakiness of the Plac promoter; even low-level MazF expression was sufficient to kill all successfully transformed host cells. This outcome demonstrated that the Plac/LacI system lacks the tight regulatory control necessary for highly toxic genes.

To overcome this critical limitation, the study transitioned to the arabinose-inducible PBAD promoter, which offers much stronger repression under non-inducing conditions. Using this system, the PBAD–MazF plasmid was successfully constructed, and a stable recombinant strain (DH5α–PBAD–MazF) was obtained. This success confirmed that the PBAD system effectively suppresses basal expression of MazF in the absence of the inducer.

Subsequent functional testing evaluated system performance through growth curve monitoring. Under non-inducing conditions (no arabinose), the engineered strain exhibited normal growth, verifying the system’s high safety and stability. Under inducing conditions (with arabinose), expression of MazF toxin was expected to cause rapid growth arrest or severe inhibition, confirming inducible self-killing capacity.

In summary, the PBAD-based MazF suicide system provides a feasible and efficient biosafety solution, enabling precise and stringent control over the fate of engineered bacteria through an externally applied inducer.

This experimental module focuses on building a kill switch system in E. coli using the bacterial toxin MazF, a stable endoribonuclease that induces cell death. The initial design attempted to use a lactose-inducible Plac promoter, but due to leaky expression and strong toxicity of MazF, no positive clones could be obtained. The strategy was later revised to employ the PBAD promoter, which is tightly regulated by arabinose, enabling inducible control of MazF expression.

Key Steps:

• Aug 4–6: Preparation of competent E. coli cells, cloning of Plac-mazF construct.
• Aug 7–8: Transformation failed due to leaky expression; switch to PBAD system.
• Aug 9–11: Cloning and successful transformation of PBAD-mazF into E. coli DH5α.
• Aug 12–13: Strain preservation and overnight activation.
• Aug 14–15: Growth curve analysis with and without arabinose induction to assess MazF toxicity.

📅 Daily Plan Summary + Experimental Protocol

Date	Task	Objective	Expected Outcome
August4	Preparation of lab materials (LB, CaCl₂, glycerol, Amp, mazF DNA), inoculate DH5α	Set up for transformation and toxin testing	Competent cell prep and starter cultures
August5	Prepare competent cells with CaCl₂, grow DH5α and BL21 to OD600 ~0.5	Generate competent E. coli for cloning experiments	Aliquoted ice-cold competent DH5α cells
August6	Extract pSB1A3 plasmid, clone Plac-mazF with LacI upstream	Build first version of kill switch under Plac control	Recombinant plasmid Plac-LacI-mazF
August7	Transform Plac-LacI-mazF into DH5α	Attempt to obtain stable transformants	❌ No colonies due to MazF leakage
August8	Troubleshooting: determine Plac leaky expression is incompatible	Conclude Plac-based design fails due to MazF toxicity	Strategy shift to PBAD promoter
August9	Clone PBAD-mazF into pSB1A3 using XbaI/SpeI and T4 ligation overnight	Construct new tightly regulated suicide switch	PBAD-MazF recombinant plasmid
August10	Transform PBAD-mazF into DH5α (heat shock, 42°C, 1 min)	Establish suicide switch strain	Colony growth on LB + Amp plates
August11	Colony PCR, sequencing verification, inoculate positive clone	Confirm correct insertion and amplify culture	DH5α-PBAD-MazF strain obtained
August12	Glycerol stock preparation, begin expansion culture	Preserve and expand the engineered strain	Frozen stocks, 5 mL liquid culture
August13	Overnight culture in LB + Amp	Pre-induction activation	Healthy pre-culture for growth assay
August14	Inoculate into fresh LB with and without 2% arabinose, measure OD600	Induce PBAD promoter to express MazF, monitor effect	Growth arrest in induced group
August15	Analyze growth curves, compare induced vs. uninduced groups	Evaluate MazF toxicity upon PBAD induction	Successful kill switch confirmed

Date: 2025-08-04

Experiment Title: Preparation of Reagents, Media, and Initial Inoculation for MazF Toxin Expression Objective:

To prepare all necessary reagents and culture conditions for construction of a bacterial suicide system using the MazF toxin gene. Start seed cultures of E. coli DH5α for competent cell preparation.

Experimenters:

Yeqiao Ma

Zhiyan Ding

Guozhi Ji

Tailai Cong

Yixuan Fan

Binqi Qian

Materials and Reagents:

• LB agar and LB broth media components
• Ampicillin stock (100 mg/mL)
• 0.1 M CaCl₂ solution (pre-cooled, sterile)
• 50% glycerol (v/v) for storage
• mazF gene sample (synthesized, lyophilized or plasmid form)
• Primers for mazF, lacI, Plac
• PCR reaction mix (2× Phanta, Vazyme or similar)
• E. coli DH5α strain
• Sterile cryovials, culture tubes, pipette tips (autoclaved)
• Shaking incubator, water bath, ice bucket

Methods:

1. Reagent and Media Preparation

Prepared 1 L LB broth and 500 mL LB agar (with 1.5% agar). Autoclaved.

Filter-sterilized 100 mL of 0.1 M CaCl₂ and stored on ice.

Prepared 20 mL of sterile 50% glycerol.

Diluted ampicillin stock to working concentration of 100 µg/mL in LB.

Rehydrated synthesized mazF gene sample with nuclease-free water and stored at –20°C.

Resuspended primers in TE buffer and diluted to 10 µM.

1. Seed Culture Preparation

Inoculated a single colony of E. coli DH5α into 5 mL LB broth.

Incubated at 37°C, 150 rpm overnight. This culture was used as the seed for competent cell prep.

Results:

• All reagents, antibiotics, and stocks were successfully prepared and clearly labeled.
• E. coli DH5α culture showed healthy turbidity (~OD600 >1.5) by next morning.

Conclusion / Next Steps:

• Proceed with competent cell preparation for transformation on 08/05.
• Prepare BL21 strain as a backup for future comparative expression.

Date: 2025-08-05

Experiment Title: Preparation of Chemically Competent E. coli DH5α and BL21 Cells Objective:

To prepare chemically competent E. coli DH5α and BL21 cells using CaCl₂ treatment for transformation of the suicide system plasmids.

Experimenters:

Shumo Feng

Zhen Liu

Jiatan Guo

Xinyi Tang

Yanjia Shao

Materials and Reagents:

• Overnight E. coli DH5α culture from 08/04
• E. coli BL21 stock
• LB broth
• 0.1 M CaCl₂ (ice-cold, sterile)
• Ice bucket
• Centrifuge (4°C)
• 50 mL sterile centrifuge tubes
• Pipettes and sterile tips

Methods:

1. Culture Growth

Inoculated 1:100 of overnight DH5α into 50 mL LB broth.

Inoculated 1:100 of BL21 into 50 mL LB broth.

Incubated both cultures at 30°C, 220 rpm until OD600 reached 0.4–0.6 (~3 h).

1. Cooling and Harvesting

Placed cultures on ice for 30 min to slow metabolism and prepare membranes for CaCl₂ uptake.

Centrifuged at 4,000 × g for 10 min at 4°C.

Carefully discarded supernatant and resuspended cell pellets in 20 mL ice-cold 0.1 M CaCl₂.

1. Incubation in CaCl₂

Incubated cell suspensions on ice for another 30 min.

Aliquoted 200 µL into sterile pre-chilled microcentrifuge tubes.

Stored on ice for immediate transformation or at –80°C for future use.

1. Parallel Plasmid Culture

Inoculated E. coli DH5α carrying pSB1A3 into 5 mL LB + Amp (100 µg/mL) for plasmid extraction on 08/06.

Results:

• Obtained ice-cold competent DH5α and BL21 cells at high efficiency.
• Growth curve showed expected OD600 range; no contamination observed.
• pSB1A3 culture turbid and ready for miniprep next day.

Conclusion / Next Steps:

• Proceed with pSB1A3 plasmid extraction and cloning of Plac-mazF into pSB1A3 on 08/06.

Date: 2025-08-06

Experiment Title: Extraction of pSB1A3 Plasmid and Construction of Plac-mazF Suicide System Objective:

To extract pSB1A3 plasmid from E. coli DH5α and clone the mazF gene under the lactose-inducible Plac promoter with lacI regulation to create a suicide system prototype.

Experimenters:

Yeqiao Ma

Tailai Cong

Guozhi Ji

Yutong Li

Zehan Song

Materials and Reagents:

• Overnight E. coli DH5α-pSB1A3 culture (from 08/05)
• Plasmid miniprep kit (TIANGEN or equivalent)
• mazF gene (synthesized)
• lacI operon PCR fragment
• Primers for mazF and lacI with overlapping sequences
• High-fidelity polymerase (Phanta Max or PrimeSTAR)
• Restriction enzymes XbaI and SpeI
• T4 DNA ligase and buffer (NEB)
• Agarose gel electrophoresis setup
• DNA gel extraction kit
• Ice, pipettes, sterile tips

Methods:

1. pSB1A3 Plasmid Extraction

Performed miniprep of 5 mL overnight culture of DH5α-pSB1A3.

Measured DNA concentration (~150 ng/µL). Stored at –20°C.

1. PCR Amplification of Insert

Amplified mazF gene placing it downstream of the Plac promoter.

Amplified lacI operon separately.

Linked lacI upstream of Plac-mazF with B0015 terminator as spacer via overlap PCR.

Expected size of PlacI-LacI-B0035-Plac-mazF fragment ~3–3.5 kb. Verified by agarose gel electrophoresis.

1. Restriction Digestion

Digested purified PCR fragment and pSB1A3 vector with XbaI and SpeI at 37°C for 2 h.

Purified digested products using DNA gel extraction kit.

1. Ligation

Set up T4 DNA ligase reaction with 3:1 insert:vector molar ratio.

Incubated at 16°C overnight for ligation.

Results:

• High-quality pSB1A3 plasmid extracted.
• PCR product for PlacI-LacI-B0035-Plac-mazF correctly amplified and visible on gel at expected size.
• Digestion completed successfully; ligation reaction set for overnight incubation.

Conclusion / Next Steps:

• Transform the ligation product into E. coli DH5α on 08/07.

Date: 2025-08-07

Experiment Title: Transformation of Plac-mazF Suicide Construct into E. coli DH5α Objective:

To transform the constructed suicide plasmid (PlacI-LacI-B0035-Plac-mazF in pSB1A3 backbone) into E. coli DH5α and screen for positive clones.

Experimenters:

Xinyi Tang

Binqi Qian

Zhiyan Ding

Zehan Song

Materials and Reagents:

• Ligation product from 08/06
• Chemically competent E. coli DH5α cells (prepared on 08/05)
• LB agar plates with 100 μg/mL Ampicillin
• SOC medium
• Water bath at 42°C
• Ice
• Sterile 1.5 mL tubes

Methods:

1. Heat-Shock Transformation

Mixed 5 µL of ligation product with 50 µL competent DH5α cells.

Incubated on ice for 30 min.

Heat shocked at 42°C for 60 seconds.

Immediately placed back on ice for 2 min.

Added 450 µL SOC medium, incubated at 37°C for 1 hour with shaking.

1. Plating

Spread 100 µL of recovery culture on LB agar plates containing 100 µg/mL Ampicillin.

Incubated overnight at 37°C.

Results:

• No colonies observed on the Ampicillin selection plates after overnight incubation.

Troubleshooting Analysis:

• It is likely that leaky expression from the Plac promoter led to basal-level mazF toxin expression, resulting in cell death prior to colony formation.
• Even with lacI present for repression, Plac is known to exhibit background expression without IPTG, which may have been enough to activate MazF toxicity.

Conclusion / Next Steps:

• Due to the toxicity of mazF under even slight expression, an inducible and tightly regulated promoter is needed.
• Plan revised: construct system using PBAD promoter, which offers tighter repression in absence of arabinose. Proceed with this design on 08/09.

Date: 2025-08-08

Experiment Title: Post-Transformation Evaluation and Failure Analysis of Plac-mazF Suicide System Objective:

To evaluate the transformation results from the Plac-mazF construct and troubleshoot the failure to recover any viable colonies.

Experimenters:

Guozhi Ji

Shumo Feng

Zhen Liu

Materials and Reagents:

• LB agar plates with 100 µg/mL Ampicillin (from 08/07)
• E. coli DH5α competent cells
• Ligation mix from 08/06
• SOC medium
• Lab notebook and previous transformation control data

Methods & Observations:

1. Colony Check

Checked transformation plates from 08/07 after >16 h at 37°C.

Confirmed no colony growth on any plates with the Plac-mazF construct.

1. Control Comparison

Positive control transformations (e.g., pSB1A3 empty vector) done in parallel showed healthy colony growth, confirming the competency of DH5α cells and quality of the LB/Amp plates.

1. HypothesisLeaky expression of Plac promoter led to low-level transcription of mazF, producing MazF toxin even in the absence of IPTG.
2. LacI repression was insufficient, particularly under multicopy plasmid context (pSB1A3 is high-copy).
3. Result: early expression of mazF during transformation or recovery phase killed host cells before colony formation.
4. Decision

Discontinue attempts to use Plac promoter for toxin expression.

Move to use the PBAD promoter, known for tight regulation in the absence of arabinose and no background leakiness.

Results:

• No colonies recovered.
• Positive control showed normal transformation efficiency, confirming that plasmid toxicity—not technical error—was the cause.

Date: 2025-08-09

Experiment Title: Construction of PBAD-MazF Suicide System Objective:

To construct a safer and tightly controlled bacterial suicide system by placing the mazF toxin gene under the arabinose-inducible PBAD promoter in the pSB1A3 backbone.

Experimenters:

Tailai Cong

Yicheng Zhang

Yeqiao Ma

Xinyi Tang

Materials and Reagents:

• Synthesized, codon-optimized mazF gene (RFC#10 compatible)
• PCR primers with flanking XbaI and SpeI restriction sites
• pSB1A3 plasmid
• High-fidelity DNA polymerase (e.g., PrimeSTAR Max)
• XbaI and SpeI restriction enzymes (NEB)
• Agarose gel electrophoresis supplies
• DNA purification and gel extraction kits
• T4 DNA ligase and buffer
• LB agar plates + 100 µg/mL Ampicillin

Methods:

1. Design & Amplification

The mazF gene was already synthesized with a PBAD promoter upstream.

The full PBAD-mazF cassette was amplified using high-fidelity PCR to ensure no mutation.

Gel electrophoresis confirmed expected size of ~1.2–1.5 kb.

1. Double Digestion

Digested both PBAD-mazF PCR product and pSB1A3 vector with XbaI and SpeI for 2 h at 37°C.

Purified the digested products using a gel extraction kit.

1. Ligation

Ligated the PBAD-mazF insert into the digested pSB1A3 vector using T4 DNA ligase at 16°C overnight.

Used a 3:1 molar ratio of insert to vector to increase ligation efficiency.

Results:

• PCR yielded high-purity PBAD-mazF fragment of correct size.
• Digestion and purification steps showed no visible contamination.
• Ligation mixture was clear and prepared for transformation the following day.

Conclusion / Next Steps:

• Transform the PBAD-mazF construct into E. coli DH5α on 08/10.
• Expect improved cloning efficiency due to low basal expression from PBAD in absence of arabinose.

Date: 2025-08-10

Experiment Title: Transformation of PBAD-MazF Suicide Construct into E. coli DH5α Objective:

To introduce the newly constructed pSB1A3-PBAD-mazF plasmid into E. coli DH5α using heat-shock transformation, enabling future functional tests of the inducible suicide system.

Experimenters:

Guozhi Ji

Zehan Song

Shumo Feng

Yixuan Fan

Materials and Reagents:

• Ligation mix from 08/09 (PBAD-mazF in pSB1A3)
• Competent E. coli DH5α cells (prepared previously)
• SOC recovery medium
• LB agar plates containing 100 µg/mL Ampicillin
• Sterile 1.5 mL microcentrifuge tubes
• Ice and water bath (42°C)

Methods:

1. Heat-Shock Transformation

Mixed 5 µL of ligation reaction with 50 µL DH5α competent cells.

Incubated on ice for 30 minutes.

Performed heat-shock at 42°C for 60 seconds.

Returned tubes to ice for 2 minutes.

Added 450 µL SOC medium and incubated at 37°C, 180 rpm for 1 hour.

1. Plating

Spread 100 µL of transformed cells on LB agar plates with Ampicillin.

Incubated plates overnight at 37°C.

Results:

• Transformation process completed without visible contamination.
• Plates incubated for evaluation on the following day (08/11).

Conclusion / Next Steps:

• Screen for colonies on 08/11.
• Sequence positive clones to confirm plasmid integrity and orientation.
• If successful, proceed to growth inhibition tests by arabinose induction (planned from 08/13 onward).

Date: 2025-08-11

Experiment Title: Screening and Validation of E. coli DH5α-PBAD-MazF Clones Objective:

To identify and verify E. coli DH5α transformants containing the PBAD-mazF suicide plasmid, and prepare verified clones for future induction experiments.

Experimenters:

Shuyi Liu

Tailai Cong

Yeqiao Ma

Yicheng Zhang

Materials and Reagents:

• LB agar plates with colonies from 08/10 transformation
• 5 mL LB + Amp (100 μg/mL) liquid cultures
• Tsingke sequencing service
• 25% glycerol for strain storage
• Sterile inoculation loops
• Shaking incubator at 37°C, 150 rpm

Methods:

1. Colony Selection & Inoculation

Selected 3–5 single colonies from LB/Amp plates.

Inoculated each into 5 mL LB + Amp medium.

Cultured at 37°C, 150 rpm overnight for plasmid miniprep and sequencing.

1. Strain Storage

Prepared glycerol stocks of promising colonies:

1. Mixed 750 µL overnight culture with 250 µL sterile 100% glycerol (final concentration 25%).
2. Stored at –20°C.
3. Plasmid Extraction & Sequencing Miniprepped plasmids and submitted for sequencing (Tsingke, Beijing) using VF2/VR primers to cover the PBAD-mazF insert.
4. Sent samples via cold chain to preserve plasmid quality.

Results:

• Overnight cultures showed good growth, suggesting low or no leakiness of PBAD promoter in uninduced conditions.
• Colonies sent for sequencing to confirm correct insert.

Conclusion / Next Steps:

• Await sequencing results to confirm the presence and accuracy of PBAD-mazF cassette.
• Once verified, proceed to functional testing (growth inhibition assays) starting 08/13.

Date: 2025-08-12

Experiment Title: Culture Expansion and Storage of Validated DH5α-PBAD-MazF Clones Objective:

To preserve and expand the E. coli DH5α strains harboring the PBAD-mazF suicide plasmid in preparation for induction testing.

Experimenters:

Zhiyan Ding

Leyao Xin

Xinyi Tang

Zhen Liu

Materials and Reagents:

• Confirmed E. coli DH5α-PBAD-mazF colonies (from 08/11)
• LB broth + 100 μg/mL Ampicillin (Servicebio, G3102)
• 25% glycerol for cryostorage
• Shaking incubator (37°C, 150 rpm)
• Sterile 15 mL and 1.5 mL tubes

Methods:

1. Liquid Culture Expansion

Inoculated 5 mL LB + Amp medium with single verified colonies from 08/11.

Cultured overnight at 37°C, 150 rpm to obtain dense, healthy cultures for upcoming testing.

Monitored turbidity and ensured consistent growth across replicates.

1. Cryopreservation

Prepared additional glycerol stocks (25% v/v) using freshly grown cultures.

Transferred 750 µL culture into 1.5 mL tubes, added 250 µL glycerol, mixed well, and stored at –20°C.

Results:

• All selected clones showed robust overnight growth in Amp-supplemented medium.
• Glycerol stocks successfully prepared for long-term use.
• Cultures ready for growth inhibition assays under PBAD induction starting 08/13.

Conclusion / Next Steps:

• Proceed to test the suicide system function by inducing MazF expression with L-arabinose and measuring the impact on bacterial growth on August 13.

Date: 2025-08-13

Experiment Title: Functional Induction Test of PBAD-MazF Suicide System Using L-Arabinose Objective:

To evaluate the effectiveness of the PBAD-mazF suicide construct in inhibiting bacterial growth upon induction with arabinose.

Experimenters:

Zehan Song

Yifan Tian

Binqi Qian

Jiatan Guo

Materials and Reagents:

• E. coli DH5α-PBAD-MazF glycerol stock (from 08/12)
• LB broth with 100 μg/mL Ampicillin
• L-Arabinose (2% final concentration, A83229, Innochem)
• FlexStation 3 microplate reader (Molecular Devices, USA)
• 96-well microplates
• Sterile PBS
• Shaking incubator (37°C, 150 rpm)

Methods:

1. Overnight Culture Revival

Inoculated DH5α-PBAD-MazF from glycerol stock into 5 mL LB + Amp.

Incubated overnight at 37°C, 150 rpm.

1. Experimental Setup

Diluted overnight culture 1:100 into fresh LB + Amp.

Adjusted culture to OD600 = 0.1.

Split into two conditions (triplicates each): a) Induced group: 2% L-Arabinose added to final volume b) Control group: No arabinose added

1. Growth Monitoring

Transferred 200 µL of each culture condition into 96-well microplate wells.

Placed into FlexStation 3.

Monitored OD600 every 15 minutes at 37°C, with continuous shaking, for up to 12 hours.

Results:

• Control group showed normal exponential growth curve, reaching stationary phase by ~8 h.

Induced group exhibited dramatic inhibition in growth:

• No significant increase in OD600 beyond ~2 h.
• Strong evidence of mazF-mediated growth arrest upon arabinose induction.
• This confirmed tight control of PBAD and functional cytotoxicity of MazF under inducible conditions.

Conclusion / Next Steps:

• PBAD-mazF suicide system is successfully functional.
• Use this strain as a chassis for further safety switch validation under co-culture and in vivo simulations.
• Document growth curve data and prepare plots for inclusion in Notebook and Presentation.

Date: 2025-08-14

Experiment Title: Analysis and Visualization of PBAD-MazF Growth Curve Data Objective:

To process, analyze, and visualize growth data from the arabinose-induced suicide system assay (08/13), and confirm functionality of the PBAD-mazF construct.

Experimenters:

Shumo Feng

Yutong Li

Yeqiao Ma

Xinyi Tang

Materials and Tools:

• Growth curve data (OD600 readings from FlexStation 3)
• Microsoft Excel / GraphPad Prism / Origin
• Fluorescence microscope (if needed for follow-up observation)
• Laptop for data processing
• DH5α-PBAD-MazF cultures (optional additional replicates)

Methods:

1. Data Extraction & Cleaning

Exported raw OD600 data from FlexStation 3.

Organized into time-series format for both control and induced groups.

Removed outliers and corrected baseline fluctuations.

1. Growth Curve Plotting

Plotted OD600 vs. time for both groups using GraphPad Prism.

Added error bars (standard deviation of triplicates) for statistical robustness.

Highlighted key timepoints of growth inhibition onset (~2 h post induction).

1. Interpretation

Confirmed that arabinose-induced MazF expression caused a significant and irreversible growth arrest in E. coli DH5α.

No growth rebound observed within 12-hour window, suggesting efficient toxin expression.

1. Optional Imaging

Briefly examined induced and uninduced cultures under light microscopy.

Induced cells appeared mostly lysed or arrested in division.

Conclusion / Next Steps:

• Final validation of the suicide switch system.
• Consider incorporating the MazF module into higher-level systems (e.g., under multi-input logic or co-culture models).
• Prepare a summarized version for internal presentation and troubleshooting guide.

Date: 2025-08-15

Experiment Title: Documentation and Presentation Preparation of PBAD-MazF Safety Module Objective:

To finalize all experimental records, figures, and analysis associated with the PBAD-MazF inducible suicide system for integration into the iGEM Notebook and presentation materials.

Experimenters:

Jiatan Guo

Guozhi Ji

Zehan Song

Yicheng Zhang

Materials and Tools:

• GraphPad Prism / Microsoft PowerPoint
• FlexStation 3 growth curve output
• Lab notebooks (physical and digital)
• Adobe Illustrator or similar tools for figure layout
• Wiki template (for integrating final results)

Methods:

1. Data Review

Cross-checked OD600 datasets with raw machine logs.

Verified culture conditions, arabinose concentration, and OD starting points were correctly recorded.

1. Figure Finalization

Exported growth curve with proper labeling, units, and annotations.

Generated separate plots for: a) Full time-course comparison b) Log-phase zoom-in for MazF-induced growth inhibition onset

Designed a composite figure showing PBAD-MazF design, experimental workflow, and growth results.

1. Notebook Entry Completion

Compiled daily entries from August 4–15.

Standardized format with: Objective, Methods, Results, and Conclusion.

Included strain table, key plasmid map, and verification sequencing summary.

1. Presentation Material Prep

Drafted slides for internal group meeting to explain suicide switch design, problems with Plac system, and success with PBAD-based strategy.

Highlighted the safety value of using tight, non-leaky PBAD control for toxic gene expression.

Results:

• Growth inhibition results clearly support MazF function and validate PBAD as a controllable safety switch.
• Materials ready for team review and publication.

Conclusion / Next Steps:

• Finalize cloning strategy if integration with additional circuits is needed.
• Submit composite figures to team graphics designer.
• Upload validated results to Registry if parts are original.