iGEM 2025 — Team rdfz-china

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Content

* [System 1: Therapeutic system - Expression, purification and activity verification of thrombin (Coa), July 1 – July 14](#section-1)
  + [Date: 2025-07-02](#subsection-2)
  + [Date: 2025-07-03](#subsection-3)
  + [Date: 2025-07-04](#subsection-4)
  + [Date: 2025-07-05](#subsection-5)
  + [Date: 2025-07-06](#subsection-6)
  + [Date: 2025-07-07](#subsection-7)
  + [Date: 2025-07-08](#subsection-8)
  + [Date: 2025-07-09](#subsection-9)
  + [Date: 2025-07-10](#subsection-10)
  + [Date: 2025-07-11](#subsection-11)
  + [Date: 2025-07-12](#subsection-12)
  + [Date: 2025-07-13](#subsection-13)
  + [Date: 2025-07-14](#subsection-14)
* [System 2: Near-Infrared (NIR) Light-Inducible Biosensor Module, July 15 – July 25](#section-15)
  + [Date: 2025-07-15](#subsection-16)
  + [Date: 2025-07-16](#subsection-17)
  + [Date: 2025-07-17](#subsection-18)
  + [Date: 2025-07-18](#subsection-19)
  + [Date: 2025-07-19](#subsection-20)
  + [Date: 2025-07-20](#subsection-21)
  + [Date: 2025-07-21](#subsection-22)
  + [Date: 2025-07-22](#subsection-23)
  + [Date: 2025-07-23](#subsection-24)
  + [Date: 2025-07-24](#subsection-25)
  + [Date: 2025-07-25](#subsection-26)
* [System 3: Colorectal Cancer (CRC)-Targeting Adhesion System, July 26-August 3](#section-27)
  + [Date: 2025-07-26](#subsection-28)
  + [Date: 2025-07-27](#subsection-29)
  + [Date: 2025-07-28](#subsection-30)
  + [Date: 2025-07-29](#subsection-31)
  + [Date: 2025-07-30](#subsection-32)
  + [Date: 2025-07-31](#subsection-33)
  + [Date: 2025-08-01](#subsection-34)
  + [Date: 2025-08-02](#subsection-35)
  + [Date: 2025-08-03](#subsection-36)
* [System 4: Construction of Bacterial Suicide System Based on Toxin MazF, August 4-August 15](#section-37)
  + [Date: 2025-08-04](#subsection-38)
  + [Date: 2025-08-05](#subsection-39)
  + [Date: 2025-08-06](#subsection-40)
  + [Date: 2025-08-07](#subsection-41)
  + [Date: 2025-08-08](#subsection-42)
  + [Date: 2025-08-09](#subsection-43)
  + [Date: 2025-08-10](#subsection-44)
  + [Date: 2025-08-11](#subsection-45)
  + [Date: 2025-08-12](#subsection-46)
  + [Date: 2025-08-13](#subsection-47)
  + [Date: 2025-08-14](#subsection-48)
  + [Date: 2025-08-15](#subsection-49)

# System 1: Therapeutic system - Expression, purification and activity verification of thrombin (Coa), July 1 – July 14

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).

![System 1: Therapeutic system - Expression, purification and activity verification of thrombin (Coa), July 1 – July 14 Diagram](https://static.igem.wiki/teams/5922/notebook/img1.webp)
![System 1: Therapeutic system - Expression, purification and activity verification of thrombin (Coa), July 1 – July 14 Diagram](https://static.igem.wiki/teams/5922/notebook/img2.webp)

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.

![Date: 2025-07-02 Diagram](https://static.igem.wiki/teams/5922/notebook/img3.webp)

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.

![Date: 2025-07-03 Diagram](https://static.igem.wiki/teams/5922/notebook/img4.webp)
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.

![Date: 2025-07-03 Diagram](https://static.igem.wiki/teams/5922/notebook/img5.webp)
![Date: 2025-07-03 Diagram](https://static.igem.wiki/teams/5922/notebook/img6.webp)
![Date: 2025-07-03 Diagram](https://static.igem.wiki/teams/5922/notebook/img7.webp)
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.

![Date: 2025-07-03 Diagram](https://static.igem.wiki/teams/5922/notebook/img8.webp)
![Date: 2025-07-03 Diagram](https://static.igem.wiki/teams/5922/notebook/img9.webp)
![Date: 2025-07-03 Diagram](https://static.igem.wiki/teams/5922/notebook/img10.webp)
![Date: 2025-07-03 Diagram](https://static.igem.wiki/teams/5922/notebook/img11.webp)
![Date: 2025-07-03 Diagram](https://static.igem.wiki/teams/5922/notebook/img12.webp)

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.

![Date: 2025-07-05 Diagram](https://static.igem.wiki/teams/5922/notebook/img13.webp)
![Date: 2025-07-05 Diagram](https://static.igem.wiki/teams/5922/notebook/img14.webp)
![Date: 2025-07-05 Diagram](https://static.igem.wiki/teams/5922/notebook/img15.webp)
![Date: 2025-07-05 Diagram](https://static.igem.wiki/teams/5922/notebook/img16.webp)

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.

![Date: 2025-07-08 Diagram](https://static.igem.wiki/teams/5922/notebook/img17.webp)
![Date: 2025-07-08 Diagram](https://static.igem.wiki/teams/5922/notebook/img18.webp)
![Date: 2025-07-08 Diagram](https://static.igem.wiki/teams/5922/notebook/img19.webp)

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.

![Date: 2025-07-11 Diagram](https://static.igem.wiki/teams/5922/notebook/img20.webp)

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.

![Date: 2025-07-14 Diagram](https://static.igem.wiki/teams/5922/notebook/img21.webp)
![Date: 2025-07-14 Diagram](https://static.igem.wiki/teams/5922/notebook/img22.webp)
![Date: 2025-07-14 Diagram](https://static.igem.wiki/teams/5922/notebook/img23.webp)

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.

# System 2: Near-Infrared (NIR) Light-Inducible Biosensor Module, July 15 – July 25

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.

![Date: 2025-07-17 Diagram](https://static.igem.wiki/teams/5922/notebook/img24.webp)
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.

![Date: 2025-07-19 Diagram](https://static.igem.wiki/teams/5922/notebook/img25.webp)
![Date: 2025-07-19 Diagram](https://static.igem.wiki/teams/5922/notebook/img26.webp)

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.

![Date: 2025-07-20 Diagram](https://static.igem.wiki/teams/5922/notebook/img27.webp)
![Date: 2025-07-20 Diagram](https://static.igem.wiki/teams/5922/notebook/img28.webp)

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

![Date: 2025-07-20 Diagram](https://static.igem.wiki/teams/5922/notebook/img29.webp)

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

![Date: 2025-07-20 Diagram](https://static.igem.wiki/teams/5922/notebook/img30.webp)
![Date: 2025-07-20 Diagram](https://static.igem.wiki/teams/5922/notebook/img31.webp)

3. The influence of infrared light on bacterial growth

![Date: 2025-07-20 Diagram](https://static.igem.wiki/teams/5922/notebook/img32.webp)

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

![Date: 2025-07-20 Diagram](https://static.igem.wiki/teams/5922/notebook/img33.webp)

5. Comparison of FC values for different chassis

![Date: 2025-07-20 Diagram](https://static.igem.wiki/teams/5922/notebook/img34.webp)

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

![Date: 2025-07-20 Diagram](https://static.igem.wiki/teams/5922/notebook/img35.webp)
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.

![Date: 2025-07-25 Diagram](https://static.igem.wiki/teams/5922/notebook/img36.webp)
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.

# System 3: Colorectal Cancer (CRC)-Targeting Adhesion System, July 26-August 3

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.

![Date: 2025-07-27 Diagram](https://static.igem.wiki/teams/5922/notebook/img37.webp)

![Date: 2025-07-27 Diagram](https://static.igem.wiki/teams/5922/notebook/img38.webp)
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.

![Date: 2025-07-27 Diagram](https://static.igem.wiki/teams/5922/notebook/img39.webp)
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.

![Date: 2025-07-28 Diagram](https://static.igem.wiki/teams/5922/notebook/img40.webp)
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.

![Date: 2025-08-01 Diagram](https://static.igem.wiki/teams/5922/notebook/img41.webp)
![Date: 2025-08-01 Diagram](https://static.igem.wiki/teams/5922/notebook/img42.webp)
![Date: 2025-08-01 Diagram](https://static.igem.wiki/teams/5922/notebook/img43.webp)
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.

![Date: 2025-08-03 Diagram](https://static.igem.wiki/teams/5922/notebook/img44.webp)
![Date: 2025-08-03 Diagram](https://static.igem.wiki/teams/5922/notebook/img45.webp)
![Date: 2025-08-03 Diagram](https://static.igem.wiki/teams/5922/notebook/img46.webp)
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.

# System 4: Construction of Bacterial Suicide System Based on Toxin MazF, August 4-August 15

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.

![Date: 2025-08-06 Diagram](https://static.igem.wiki/teams/5922/notebook/img47.webp)
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.

![Date: 2025-08-09 Diagram](https://static.igem.wiki/teams/5922/notebook/img48.webp)
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.

![Date: 2025-08-15 Diagram](https://static.igem.wiki/teams/5922/notebook/img49.webp)
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.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block page\_content %}

Content

* [Overview](#section-1)
  + [Cycle 1 NETMAP-mRFP](#section-2)
    - [Design](#subsection-3)
    - [Build](#subsection-4)
    - [Test](#subsection-5)
    - [Learn](#subsection-6)
  + [Cycle 2-1 PD-L1 nb](#section-7)
    - [Design](#subsection-8)
    - [Build](#subsection-9)
    - [Test](#subsection-10)
    - [Learn](#subsection-11)
  + [Cycle 2-2 YopE1-15-PD-L1 nb](#section-12)
    - [Design](#subsection-13)
    - [Build](#subsection-14)
    - [Test](#subsection-15)
    - [Learn](#subsection-16)
  + [Cycle 2-3 Coagulase](#section-17)
    - [Design](#subsection-18)
    - [Build](#subsection-19)
    - [Test](#subsection-20)
    - [Learn](#subsection-21)
  + [Cycle 3 INP-HlpA](#section-22)
    - [Design](#subsection-23)
    - [Build](#subsection-24)
    - [Test](#subsection-25)
    - [Learn](#subsection-26)
  + [Cycle 4-1 MazF](#section-27)
    - [Design](#subsection-28)
    - [Build](#subsection-29)
    - [Test](#subsection-30)
    - [Learn](#subsection-31)
  + [Cycle 4-2 PBAD−MazF](#section-32)
    - [Design](#subsection-33)
    - [Build](#subsection-34)
    - [Test](#subsection-35)
    - [Learn](#subsection-36)
  + [Reference](#section-37)

# Overview

This project aims to develop a precise, red light–inducible, tumor-targeted probiotic therapeutic platform for colorectal cancer (CRC). By integrating multiple engineered modules — including the NETMAP optogenetic control system, PD-L1 nanobody for immune checkpoint blockade, Coagulase for localized vascular occlusion, INP-HlpA anchoring system for tumor-specific adhesion, and the PBAD–MazF safety module for biosafety and dormancy control — we constructed a comprehensive, modular bacterial therapy system.

Each cycle of the design progressively refined a key functional component:

* Cycle 1 (NETMAP–mRFP) established a reliable red light–controlled gene expression system using 660 nm light.
* Cycle 2 (PD-L1 nb / Coagulase) implemented immune activation and vascular blockade modules for dual therapeutic action.
* Cycle 3 (INP–HlpA) enabled tumor-specific localization and adhesion to colorectal cancer cells.
* Cycle 4 (MazF/P\_\_\_SUB\_\_\_BAD\_\_\_) introduced a robust biosafety mechanism allowing dormancy and inducible suicide control.

![Overview Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img1.webp)

Figure 1. Engineering success cycles of the RectomeDy FotoZymogen system, illustrating 7 iterative Design–Build–Test–Learn (DBTL) loops

Together, these modules form a next-generation spatiotemporally controllable bacterial therapy, achieving precise activation at tumor sites, minimal off-target effects, and enhanced clinical safety - laying the groundwork for intelligent, light-controlled cancer biotherapeutics.

# Cycle 1 NETMAP-mRFP

## Design

Colorectal cancer (CRC) is the second most common cancer in China, and current treatment methods lack precision and controllability. Inspired by a recent 2024 Nature article titled “Engineered bacteria for near-infrared light-inducible expression of cancer therapeutics”, we recognized the potential of red light to precisely control the release of cancer therapeutics. This insight led us to develop a system based on NETMAP (Nano Engineered Temporal Modulation Activation Platform), which can be activated by red light. The system is designed to allow controlled therapeutic expression, triggered exclusively by red light exposure, ensuring spatial and temporal precision in therapeutic delivery.

Upon exposure to red light, PadC, a photosensitive protein, absorbs the light and undergoes a conformational change. This change facilitates the conversion of intracellular GTP into the second messenger c-di-GMP, effectively turning the light signal into a chemical signal. As the concentration of c-di-GMP rises, it activates the c-di-GMP response protein MrkH, which binds to the downstream specific promoter PMrkA. This, in turn, drives the expression of therapeutic genes, allowing precise control over the timing and location of cancer therapeutics release. This optogenetic mechanism enables non-invasive, light-controlled therapeutic applications with minimal off-target effects.

![Design Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img2.webp)

Figure 2. Mechanism of action of the NETMAP System

## Build

The NIR light-induced promoter NETMAP was synthesized by gene synthesis (Generalbiol, China). The NETMAP system consists of two plasmids. Plasmid A, based on the pSB4C5 backbone, contains chloramphenicol resistance, constitutively expresses YhjH and MrkH using the tac promoter, and carries PmrkA-mRFP. Plasmid B, based on the pSB1A3 backbone, contains ampicillin resistance and constitutively expresses PadC4 and BphO using the lac promoter. The above-synthesized sequences were cloned upstream of the mRFP coding sequence in the pSB1A3 vector via the one-step cloning method (Seamless Cloning Kit, D7010, beyotime). Subsequently, the recombinant plasmids were transformed into *E. coli* DH5α and BL21 by the heat shock method (42°C, 1 min). Positive clones were screened on LB (Luria Bertani) solid plates (supplemented with 1.5% agar) containing 100 μg/mL ampicillin (Amp) and 30 μg/mL chloramphenicol (Cm), and verified by sequencing (Tsingke, Beijing), resulting in the acquisition of recombinant engineered strains DH5α-NETMAP-mRFP and BL21-NETMAP-mRFP. The strains were stored at -20°C with 25% (v/v) glycerol used as an antifreeze agent. The engineered strains were cultured at 37°C with shaking at 150 rpm. Inoculation and expansion culture were performed in LB broth (G3102, Servicebio, China) containing 100 μg/mL Amp.

![Build  Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img3.webp)

Figure 3: Construction of NETMAP (A1) The plasmid map of pSB4C5-YhjH-MrkH. (A2) The plasmid map of pSB1A3-PadC4-BphO. (A3) The plasmid map of NETMAP. (B) Agarose gel electrophoresis of BphO (591 bp), PadC4 (2037 bp), MrkH (705 bp), and YhjH (768 bp). (C) The gene circuit of NETMAP.

## Test

We chose 660 nm red light because it is a more common wavelength for LED lights, making it more cost-effective and easier to implement in hardware, although the reference literature used 710 nm. Using 660 nm avoids the need for custom LED lights, reducing both the experimental timeline and overall hardware costs.

### Response of the NETMAP Promoter to 660 nm NIR Light

The experiment began with the optimization of the light irradiation environment (as shown in Figures A and B). Initially, an attempt was made to use an LED for light irradiation inside a clean bench (Figure A). However, due to stray light interference in the environment, obstruction of light by the test tube rack, and difficulty in maintaining a constant temperature of 37°C, the initial induction effect was unsatisfactory. Therefore, an optimized method was adopted: the LED light source was fixed inside a closed incubator (Figure B) to ensure complete isolation from external stray light. The uniformity and utilization efficiency of light were enhanced through the reflection of the stainless-steel inner wall, while the culture temperature was precisely controlled at 37°C, ensuring that all subsequent functional verification experiments were conducted under optimal and stable conditions.

After establishing a stable environment, functional verification was initiated: the engineered strain carrying the NETMAP-mRFP gene circuit (BL21-NETMAP-mRFP) was inoculated at a ratio of 1:100 into 5 mL of LB liquid medium containing 100 μg/mL Amp, and activated and cultured overnight (12 hours) in a shaker at 37°C and 180 rpm. To ensure the accuracy of subsequent experimental controls, all culture tubes were wrapped with tin foil to achieve complete light shielding. On the next day, the activated bacterial culture was transferred at a 1% inoculation rate into 500 μL of fresh Amp⁺ LB medium in a 48-well plate, followed by continued culture at 37°C and 180 rpm until the bacterial density reached an OD600 of approximately 0.5. Once the predetermined OD value was achieved, the samples in the experimental group were transferred to a closed incubator and irradiated with a 660 nm NIR light LED for 3 hours. The samples in the dark control group were continuously cultured under complete light-shielded conditions. After the induction period, bacterial samples from both groups were collected simultaneously. A microplate reader was used to measure the OD600 and mRFP fluorescence intensity (excitation wavelength: 584 nm, emission wavelength: 607 nm) of the bacteria synchronously. Subsequently, the normalized fluorescence intensity (fluorescence intensity divided by OD600) was calculated to eliminate potential differences in bacterial density among different samples, ensuring accurate and fair quantitative comparison of the actual induction efficiency of the NETMAP promoter.

![Test Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img4.webp)

Figure 4: Experimental Optimization of the NIR Light-Induced System. (A) Schematic diagram of light irradiation for the NETMAP system in the initial clean bench environment. (B) Optimized light irradiation environment for the NETMAP system in a closed incubator.

We first compared the growth of the BL21-NETMAP-mRFP strain under dark and 660 nm red light induction conditions, using OD600 as an indicator. The results indicated no significant difference in OD600 values between the two groups, suggesting that 660 nm near-infrared light had no measurable impact on bacterial growth rate. This finding rules out the possibility of near-infrared light exerting inhibitory toxicity on bacterial growth.

![Test Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img5.webp)

Figure 5: Effect of NIR on bacterial growth

We further compared the fluorescence intensity of the engineered bacteria under dark and red light conditions. The fluorescence intensity was calculated as fluorescence value/OD600, with units in A.U. The results showed that the fluorescence intensity in the red light-induced group was approximately 24.5 A.U., significantly higher than the 5.8 A.U. observed under dark conditions. This demonstrates that 660 nm near-infrared light effectively activates the NETMAP promoter, inducing gene expression.

![Test Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img6.webp)

Figure 6: Fluorescence intensity of BL21-NETMAP-mRFP under dark and NIR conditions

### Flow Cytometric Analysis of Single-Cell Fluorescence Distribution

The engineered strain BL21-NETMAP-mRFP was cultured until the OD600 reached approximately 0.5, after which one group was placed in the dark and the other was continuously irradiated with 660 nm NIR light for 3 hours. Referring to standard flow cytometry techniques, after the induction, 100 μL of the culture was taken and transferred to a 96-well plate or flow tube for dilution and sample preparation. Subsequently, a flow cytometer (FlexStation 3, Molecular Devices, USA) was used for single-cell fluorescence detection to assess the fluorescence changes of the engineered strain under dark conditions and 660 nm light excitation. Data were collected and analyzed via the PC5.5-A channel (the mRFP fluorescence channel, corresponding to signals near the emission wavelength of 607 nm), and the fluorescence distribution characteristics of the cell population were ultimately presented in the form of a fluorescence intensity distribution histogram.

The flow cytometric analysis chart (as shown in the figure) clearly illustrates the fluorescence intensity distribution of the cell population. The fluorescence distribution of the dark control group (gray peak) was concentrated in the region of low fluorescence intensity, indicating an extremely low basal expression level of mRFP in the absence of light induction. In contrast, the fluorescence distribution of the 660 nm NIR light-induced group (red peak) showed a significant and uniform shift toward the right (high fluorescence intensity). Flow cytometric analysis confirmed the excellent quality of the NETMAP light-controlled gene switch. 660 nm NIR light not only increased the average expression level of mRFP but also achieved efficient and uniform induction of the cell population. Light irradiation enabled the entire cell population to uniformly transition from a low-expression "off" state to a high-expression "on" state, indicating that the NETMAP system exhibits good induction stability and low biological noise at the single-cell level.

![Test Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img7.webp)

Figure 7: Flow cytometric analysis of fluorescence distribution of BL21-NETMAP-mRFP in the dark and under NIR

### Effect of Induction Time on the Dynamic Response of the NETMAP System

The engineered strain BL21-NETMAP-mRFP was cultured until the OD600 reached approximately 0.5. One group was placed in a closed incubator and irradiated with a 660 nm LED NIR light, while the other group served as the dark control. A light irradiation time gradient ranging from 0 to 8 hours was set. At predetermined time points (e.g., 1 h, 3 h, 5 h, 8 h), samples were collected simultaneously from the light-irradiated group and the dark control group. After sampling, a microplate reader was used to measure the OD600 and mRFP fluorescence intensity of the bacteria synchronously. All data were calculated as normalized fluorescence intensity to analyze the change curve of mRFP expression over time. The maximum induction time was set to 8 hours, considering the avoidance of fluorescence quenching of the reporter protein mRFP under prolonged red light irradiation.

The experimental results clearly demonstrate the dynamic response of the NETMAP system. The normalized fluorescence intensity of the dark control group remained extremely low throughout the 8-hour period, further confirming the system’s excellent low-background characteristic. In contrast, the normalized fluorescence intensity of the 660 nm light-irradiated group increased rapidly over time, with expression levels continuously rising during the initial 0 to 5 hours. The fluorescence intensity reached its peak at 5 hours, with a normalized fluorescence intensity of approximately 32.4 A.U. After 5 hours, the expression level slightly decreased, which may be attributed to the degradation of the reporter protein, fluorescence quenching, or increased cellular metabolic stress. These results show that the NETMAP system exhibits a clear time-dependent response to 660 nm red light, with a controllable induction process. The expression level of the mRFP reporter protein accumulates with prolonged light exposure, and the optimal induction time is determined to be 5 hours, at which the maximum normalized fluorescence output is achieved.

![Test Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img8.webp)

Figure 8: Relationship between induction time and fluorescence Intensity of the BL21-NETMAP-mRFP

### Effect of Chassis Strains on the Performance of the NETMAP NIR Light-Induced System

The recombinant plasmid carrying the NETMAP-mRFP gene was transformed into E. coli DH5α and BL21 strains, respectively. Both engineered strains were induced when the OD600 reached approximately 0.5 and simultaneously irradiated with 660 nm NIR light for 3 hours in a closed, constant-temperature environment, with respective dark control groups set up. After induction, a microplate reader was used to measure the OD600 and mRFP fluorescence intensity of all samples. Data processing included: calculating the normalized fluorescence intensity of each sample to evaluate the absolute expression intensity; and calculating the induction fold (normalized fluorescence intensity of the light-irradiated group / normalized fluorescence intensity of the dark control group) of each strain to evaluate the switch sensitivity.

When comparing the responses of the two chassis strains to the NETMAP system, two key parameters were analyzed: absolute output intensity and induction fold (FC).

As for the absolute output intensity，after 3 hours of 660 nm red light irradiation, the normalized fluorescence intensity of the BL21-NETMAP-mRFP strain in the light-irradiated group (approximately 24.5 A.U.) was higher than that of the DH5α-NETMAP-mRFP strain (approximately 16.5 A.U.), indicating that BL21 has a advantage in the fluorescence intensity. However, this advantage is accompanied by a significant drawback: under dark conditions, the basal background leakage of the BL21 strain (approximately 13.6 A.U.) was much higher than that of the DH5α strain (approximately 3.7 A.U.). This high background leakage directly affects the quality of the “switch” and lowers the overall performance of the system.

![Test Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img9.webp)

Figure 9: Comparison of normalized fluorescence intensity of different chassis strains in the dark and under 660 nm NIR

The comparison of induction folds further highlighted the difference between the two strains. The DH5α strain exhibited a significantly higher induction fold (approximately 4.5-fold) compared to the BL21 strain (approximately 1.8-fold). This difference is primarily attributed to the extremely low background leakage in DH5α, leading to a higher ratio of light-irradiated to dark conditions, thus improving the signal-to-noise ratio and demonstrating superior switch sensitivity. Consequently, the selection of chassis strain plays a crucial role in the performance of the NETMAP system. Given the system’s need for precise control, the DH5α strain is better suited as a chassis strain due to its enhanced switch sensitivity and lower basal leakage.

![Test Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img10.webp)

Figure 10: Comparison of fluorescence induction folds (FC) of different chassis strains

## Learn

Building on the insights gained from the optimization of the NETMAP system, we concluded that careful selection of chassis strains and optimization of experimental conditions are critical for maximizing the system’s performance. The DH5α strain, with its low background leakage and high switch sensitivity, was identified as the ideal chassis for achieving precise and efficient control of gene expression. Additionally, we determined that the optimal induction time for the NETMAP system is 5 hours, at which the maximum fluorescence output is achieved without significant degradation of the reporter protein. The 660 nm red light was found to effectively activate the NETMAP system, providing a reliable, non-invasive method for controlling gene expression. These findings have laid a solid foundation for further optimizing the system for therapeutic applications, particularly for drug delivery in colorectal cancer (CRC) therapy. The next step will focus on leveraging these insights to design and test drug-loaded bacteria capable of responding to red light-induced signals for precise and controlled release of cancer therapeutics.

# Cycle 2-1 PD-L1 nb

## Design

The PD-L1 nanobody (PD-L1 nb) is a single-domain antibody fragment derived from camelid heavy-chain antibodies. It specifically targets the Programmed Cell Death Protein 1 (PD-1) receptor on immune cells and its ligand, PD-L1, which is often upregulated in various types of cancer, including colorectal cancer (CRC). PD-L1 plays a crucial role in tumor immune evasion by binding to PD-1, effectively inhibiting T cell activation and allowing the tumor to escape immune surveillance. Therefore, targeting PD-L1 with a nanobody is an attractive therapeutic strategy to enhance anti-tumor immunity by blocking this immune checkpoint. PD-L1 nanobodies are advantageous due to their small size, high specificity, and stability, making them ideal for targeted therapy. Their small size allows for deeper tissue penetration and more efficient targeting of tumors, while their stability ensures longer shelf life and improved performance in therapeutic applications. In this cycle, we aim to design and construct a system that expresses PD-L1 nanobodies in engineered probiotics, which can be triggered by red light for precise and controlled release, allowing for localized and non-invasive cancer immunotherapy.

![Design Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img11.webp)

Figure 11: Mechanism of action of PD-L1 nanobody

## Build

Codon optimization was performed on the PD-L1 nb-encoding gene for E. coli, and restriction enzyme sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI) were removed to meet the RFC#10 standards and pET28a (m) cloning requirements. Subsequently, the target gene was cloned into the pET28a (m) vector via the NdeI and XhoI restriction enzyme sites. The recombinant plasmid was transformed into competent E. coli BL21 strain by the heat shock method (42°C, 1 min). Transformants were screened on LB solid plates containing 100 μg/mL kanamycin (Kana). After verification of positive clones by sequencing, the recombinant engineered strain BL21-PD-L1 nb was obtained and stored in a culture medium containing 25% (v/v) glycerol (-20°C).

![Build  Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img12.webp)

Figure 12: Construction of the PD-L1 nb System and Gene Circuit. (A) Plasmid map. The figure shows the plasmid map of the gene circuit used for PD-L1 nb expression. (B) Schematic diagram of the gene circuit of the PD-L1 nb system.

## Test

Induced Expression and Collection of Proteins: The bacterial culture frozen at -80°C was inoculated at a ratio of 1:100 into a centrifuge tube containing 5 mL of LB broth (supplemented with 100 μg/mL Kana) and cultured overnight at 37°C with shaking at 150 rpm. Subsequently, 1% (v/v) of the culture was re-inoculated into 30 mL of fresh LB broth and continuously cultured at 37°C with shaking at 180 rpm for 1-2 hours. When the OD600 reached 0.2, 0.5 mM IPTG was added to the medium to induce the expression of the target protein. The culture temperature was then adjusted to 16°C, and cultivation was continued for 20 hours to promote the formation of soluble proteins.

Extraction and Quantification of Intracellular Proteins: 5 mL of the fermentation broth was centrifuged (10,000×g, 5 min), and the bacterial pellet was resuspended in PBS. Ultrasonic disruption was performed on ice using an ultrasonic disruptor (ultrasonication for 1 s, interval of 3 s, 70 W, 20 min). The sonicated lysate was centrifuged at 10,000×g for 30 min at 4°C, and the supernatant was collected as the intracellular protein sample. The protein concentration was determined using the Bradford kit (P0006).

Western Blot (WB) Verification: The intracellular protein samples were separated using a 12.5% SDS-PAGE gel prepared with the one-step PAGE gel rapid preparation kit (PG113). The separated proteins were transferred to a 0.22 μm PVDF membrane via the wet transfer method. The membrane was blocked with protein-free rapid blocking solution (ED0024) at room temperature for 20 min, followed by incubation with a 1:1000-diluted mouse His-tag antibody (AH367) (primary antibody) at 4°C overnight. Finally, the membrane was incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (H+L) (A0216) (secondary antibody) at room temperature for 1 hour. Chemiluminescence analysis was performed using a protein blotting imaging system.

Western Blot analysis results showed that after IPTG induction and low-temperature cultivation for 20 hours, a clear specific band was successfully detected in the intracellular protein sample of the BL21-PD-L1 nb strain. The molecular weight of this band was consistent with the expected molecular weight of PD-L1 nb (between approximately 15-20 kDa) and was specifically recognized by the His-tag antibody. This confirms the successful expression of the target gene.

![Test Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img13.webp)

Figure 13: Protein expression of PD-L1 Nanobody

## Learn

From the expression and verification of the PD-L1 nanobody in the BL21 strain, we concluded that the target protein was successfully expressed, as confirmed by Western Blot analysis, with the molecular weight matching the expected range of 15-20 kDa. This indicates that the gene for PD-L1 nb was efficiently expressed in the engineered strain.

However, during the expression process, we observed a relatively low amount of the PD-L1 nanobody in the extracellular space. While some studies have suggested that PD-L1 can be delivered without a signal peptide, our experiments showed that the extracellular secretion of the protein was insufficient. Based on this observation and previous research(Gao et al., 2024), we decided to incorporate a YopE1-15 signal peptide into the design, as suggested in the literature. This modification will be tested in the next cycle to enhance the secretion of the PD-L1 nanobody for more efficient therapeutic applications.

# Cycle 2-2 YopE1-15-PD-L1 nb

## Design

In this cycle, we focus on enhancing the extracellular secretion of the PD-L1 nanobody by incorporating the YopE1-15 signal peptide into the gene construct. The YopE1-15 signal peptide is a short peptide derived from the Yersinia pestis YopE protein, which is well-known for its role in facilitating the secretion of proteins across bacterial membranes. The signal peptide is typically used to direct the target protein to the Sec pathway, leading to its secretion into the extracellular space.

The addition of the YopE1-15 signal peptide to the PD-L1 nanobody gene aims to improve the yield of the nanobody in the extracellular environment, allowing for easier collection and purification of the protein. This strategy is based on its well-established use in promoting protein secretion in E. coli and has been successfully implemented in several other systems. By fusing the YopE1-15 signal peptide to the N-terminus of the PD-L1 nanobody, we expect to enhance the overall efficiency of the system, making it more suitable for large-scale therapeutic applications.

![Design Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img14.webp)

Figure 14: Mechanism of action of the NETMAP-YopE1-15-PD-L1 nb

## Build

First, the sequence encoding YopE1-15-fused PD-L1 nb was synthesized by gene synthesis (Generalbiol, China). This sequence was codon-optimized for E. coli, and common BioBrick restriction enzyme sites (EcoR I, Xba I, Spe I, and Pst I) were removed to ensure compliance with RFC#10 standards and facilitate subsequent cloning. Subsequently, the one-step cloning method (Seamless Cloning Kit, D7010, beyotime) was used to replace the mRFP reporter gene in the previously constructed NIR light-induced biosensor with this YopE1-15-PDL1 nb sequence, thereby placing the expression of PDL1 nb under the control of the NETMAP promoter. The obtained recombinant plasmid was transformed into the preferred chassis strain E. coli DH5α by the heat shock method (42°C, 1 min). Transformants were screened on LB solid plates containing 100 μg/mL ampicillin (Amp) and 30 μg/mL chloramphenicol (Cm). Positive clones were finally confirmed by sequencing verification (Tsingke). The obtained DH5α-NETMAP-PDL1 nb engineered strain was stored at -20°C with 25% (v/v) glycerol and inoculated and expanded at 37°C with shaking at 150 rpm.

![Build  Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img15.webp)

Figure 15: Construction of the PD-L1 Nanobody Expression System. (A) Plasmid map. The figure shows the plasmid map of the gene circuit used for PD-L1 nanobody (nb) expression. The core regulatory elements of the NETMAP system are all integrated into the pSB1A3 backbone. The reporter gene has been replaced with the sequence of YopE1-15-fused PD-L1 nb, whose expression is regulated by the PmrkA promoter. (B) Agarose gel electrophoresis of the YopE1-15-PD-L1 nb gene fragment.

## Test

Strain Activation and Cultivation: The DH5α-NETMAP-PDL1 nb engineered strain was inoculated at a ratio of 1:100 into 5 mL of LB medium containing 100 μg/mL Amp. A 15 mL centrifuge tube was wrapped with tin foil, and the strain was cultured at 37°C with shaking at 180 rpm for 12 hours. Subsequently, the bacterial cells were inoculated at a 1% rate into a 48-well plate containing 500 μL of Amp⁺ Cm⁺ LB medium and continuously cultured at 37°C with shaking at 180 rpm until the OD600 reached 0.5.

NIR Light Induction and Protein Sample Preparation: When the OD600 of the bacterial culture reached 0.5, a light-emitting diode (LED) lamp was used to initiate NIR light (660 nm) irradiation of the samples for 5 hours. Immediately after the induction, the supernatant of the engineered bacterial culture was collected. The supernatant was centrifuged at 12,000×g for 10 min at 4°C and then filtered through a 0.22 μm filter membrane to remove cells. Subsequently, the BeyoGold™ ultrafiltration tube (15 mL, 5 kDa MWCO, PES, FUF505, Beyotime) was used for protein concentration by centrifugation at 4,000×g for 40 min at 4°C. The protein concentration of the concentrated protein sample was determined using the Bradford kit (P0006, Beyotime).

Western Blot (WB) Analysis: The concentrated supernatant protein samples were separated by electrophoresis using a 12.5% SDS-PAGE gel prepared with the one-step PAGE gel rapid preparation kit (PG113, Yamei). Subsequently, the proteins were transferred to a 0.22 μm PVDF membrane (WJ001S, Yamei) via the wet transfer method. The membrane was blocked with protein-free rapid blocking solution (ED0024, Sikejie) at room temperature for 20 min. After blocking, the membrane was incubated with a 1:1000-diluted mouse His-tag antibody (AH367, Beyotime) (primary antibody) at 4°C overnight. Finally, the membrane was incubated with HRP-labeled goat anti-mouse IgG (H+L) (A0216, Beyotime) (secondary antibody) at room temperature for 1 hour. Chemiluminescence analysis was performed using a protein blotting imaging system (ChemiDoc MP, BioRad).

Western Blot (WB) analysis results showed that after 5 hours of induction with 660 nm NIR light, a clear specific band was successfully detected in the concentrated supernatant sample of the DH5α-NETMAP-PDL1 nb experimental group. The molecular weight of this band was consistent with the expected size of YopE1-15-PD-L1 nb（approximately 18 kDa）.

![Test Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img16.webp)

Figure 16: WB experimental results of YopE1-15-PD-L1 nb

## Learn

Through the construction and expression of YopE1-15-fused PD-L1 nanobody, we successfully enhanced the extracellular secretion of the PD-L1 nanobody, as confirmed by Western Blot analysis. The molecular weight of the detected band matched the expected size of approximately 18 kDa, indicating successful protein expression. This was an important achievement as it showed that the YopE1-15 signal peptide effectively promoted the secretion of the nanobody.

However, we observed that the extracellular yield, although improved, was still not as high as desired. This indicates that further optimization is required, particularly in enhancing secretion efficiency for large-scale therapeutic applications. In the next step, we plan to incorporate Coagulase (Coa), a therapeutic protein, into the system, to explore its potential as an additional payload for targeted cancer therapy.

# Cycle 2-3 Coagulase

## Design

Coagulase (Coa) is an enzyme capable of converting fibrinogen into fibrin, thereby inducing localized blood coagulation. This reaction results in the formation of fibrin clots, which block local blood flow. In cancer therapy, such localized coagulation mechanisms have emerged as a novel strategy to selectively block tumor vasculature, leading to the interruption of oxygen and nutrient supply and causing ischemic necrosis of tumor tissue, effectively “starving” tumor cells. In addition, the localized coagulation process forms a natural biological barrier that prevents engineered bacteria or therapeutic molecules from entering the bloodstream, further improving treatment precision and biosafety.

![Design Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img17.webp)

Figure 17: Mechanism of action of coagulase (Coa)

The Coa used in this project is a truncated and optimized protein fragment that enables efficient expression in E. coli while maintaining complete coagulation activity. Unlike the full-length version, this engineered fragment does not induce angiogenesis, ensuring a higher level of biosafety for therapeutic applications. Moreover, Coa can be effectively secreted through the natural secretion pathway of E. coli into the extracellular tumor microenvironment. In this cycle, we will independently verify the coagulation activity of Coa, evaluating its potential in localized vascular occlusion and tumor microenvironment modulation. In the future, we plan to integrate Coa with the NETMAP red light-inducible system and the PD-L1 nanobody module, aiming to establish a precision colorectal cancer therapy that combines optogenetic immune activation with localized tumor blockade.

![Design Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img18.webp)

Figure 18: Schematic illustration of the ideal therapeutic mechanism combining red light induction, PD-L1 nanobody activation, and coagulase-mediated vascular blockade

## Build

After codon optimization of the Coagulase-encoding gene and removal of restriction enzyme sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI), the gene was cloned into the pET28a (m) vector via the NdeI and XhoI restriction enzyme sites. The recombinant plasmid was transformed into competent E. coli BL21 strain by the heat shock method (42°C, 1 min). Transformants were screened on LB solid plates containing 100 μg/mL kanamycin (Kana) to obtain the recombinant engineered strain BL21-Coa.

![Build  Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img19.webp)

Figure 19: Construction of the Coagulase Expression System and Gene Circuit. (A) Plasmid map. The figure shows the plasmid map of the gene circuit used for Coa expression. (B) Agarose gel electrophoresis of the Coa gene fragment. (C) Schematic diagram of the Coa system gene circuit.

## Test

### Verification of Coagulase Expression by Western Blotting

After overnight culture of the BL21-Coa strain in LB broth containing Kana, it was inoculated at a ratio of 1:100 into 10 mL of fresh medium and cultured at 37°C with shaking at 180 rpm until the OD600 reached 0.5-0.8. At this point, 0.5 mM IPTG inducer was added, and the culture temperature was adjusted to 16°C, with cultivation continued for 20 hours. After fermentation, the supernatant was discarded by centrifugation (5,000×g, 10 min), and the bacterial pellet was resuspended in 10 mL of PBS. Ultrasonic disruption was performed on ice using an ultrasonic disruptor (ultrasonic power: 70%; ultrasonic cycle: 1 s on, 3 s off; total ultrasonic time: approximately 15 min). Subsequently, centrifugation was conducted at 5,000×g for 10 min at 4°C, and the supernatant was collected as the crude protein sample. The concentration was determined using the Bradford kit. For Coa protein purification and WB verification: The protein purification kit (Beyotime, P2226) was used for nickel column affinity chromatography (Ni-NTA) purification to obtain purified Coa protein. The collected purified protein fractions were analyzed for purity by SDS-PAGE, and the expression of the target protein was verified by Western Blot: The protein samples were separated using a 12.5% SDS-PAGE gel prepared with the one-step PAGE gel rapid preparation kit. The proteins were transferred to a PVDF membrane, and after blocking with protein-free rapid blocking solution, the membrane was sequentially incubated with a 1:1000-diluted mouse His-tag antibody (primary antibody) and HRP-labeled goat anti-mouse IgG secondary antibody. Finally, chemiluminescence analysis was performed using a protein blotting imaging system. The concentration of the final purified protein was determined using the Bradford kit.

SDS-PAGE and Western Blot results showed that after IPTG induction, low-temperature cultivation, and nickel column affinity chromatography, the purified Coa protein exhibited a clear and single main band on the gel, consistent with the expected molecular weight (approximately 40 - 55 kDa). WB results confirmed the successful expression and purification of the target protein.

![Test Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img20.webp)

Figure 20: WB experimental results of Coa-his tag

### Coagulase Coagulation Activity

Non-anticoagulated blood samples were used. Subsequently, 50 μL of purified Coa protein solutions with different concentrations were mixed with 50 μL of blood samples in EP tubes or 96-well plates, with a total reaction volume of 100 μL. The concentration gradient of the Coa protein solution included 100%, 80%, 60%, 40%, and 20%, and a mixture of 0% Coa solution (i.e., buffer) and 50 μL of blood sample was used as the negative control. Meanwhile, 100 μL of water and 100 μL of 100% Coa solution were set as blank controls without blood. All mixtures were incubated in a 37°C incubator, and photos were taken every 5 minutes to record the coagulation status for 30 minutes. A final photo was taken at 60 minutes when complete coagulation was achieved. The main observation and analysis indicators included thrombosis formation time, thrombus morphology, and coagulation intensity.

The Coa protein exhibited concentration-dependent coagulation activity: the 100% and 80% concentration groups showed the fastest coagulation rate, with stable thrombus formation expected within 5-10 minutes and the highest coagulation intensity. As the Coa concentration decreased gradiently, the coagulation time was significantly prolonged. The coagulation rate and intensity of the negative control group (0% Coa) were much lower than those of the Coa experimental groups. The in vitro coagulation assay results confirmed that the purified Coagulase protein has coagulation activity, and its coagulation effect is positively correlated with the protein concentration.

![Test Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img21.webp)

Figure 21: Coagulation Experiment

## Learn

Through the expression, purification, and functional verification of the Coagulase (Coa) protein, we successfully confirmed that the truncated Coa fragment can be efficiently expressed in *E. coli* and retains its biological coagulation activity. The SDS-PAGE and Western Blot analyses verified the correct expression and purity of the target protein, while the in vitro coagulation experiments demonstrated that Coa exhibits clear concentration-dependent clotting ability, forming stable thrombi within minutes at higher concentrations.

These results indicate that the engineered Coa fragment maintains its functional integrity and provides a solid foundation for its integration into subsequent NETMAP red light–inducible therapeutic systems. In future work, we will combine Coa with the PD-L1 nanobody module to construct a dual-function therapeutic platform capable of both immune checkpoint blockade and tumor vascular occlusion, aiming to achieve a more effective and localized colorectal cancer treatment strategy.

# Cycle 3 INP-HlpA

## Design

To achieve precise spatial localization of engineered bacteria within colorectal cancer (CRC) tissues, we designed a tumor-targeting anchoring system based on the fusion of HlpA (Histone-like Protein A) and INP (Ice Nucleation Protein). CRC cells are characterized by the high surface expression of heparan sulfate proteoglycans (HSPGs), adhesive glycoconjugates that play a key role in tumor cell adhesion and extracellular matrix (ECM) interactions. Leveraging this biological feature, we selected HlpA, a well-characterized heparin-binding protein with strong affinity toward HSPGs. This enables engineered bacteria to achieve specific adhesion to tumor tissues through biochemical recognition rather than passive accumulation, thereby enhancing targeting efficiency and localization accuracy.

To ensure that HlpA is properly exposed on the bacterial surface, we fused it to the outer membrane-anchoring domain of INP, an ice nucleation protein that enables the surface display of heterologous proteins without compromising bacterial viability. The resulting INP-HlpA fusion protein is stably expressed on the *E. coli* outer membrane, equipping the bacteria with tumor-specific recognition and adhesion capabilities. This fusion structure enhances local bacterial retention within the CRC microenvironment, reduces systemic spread, and increases the concentration of therapeutic molecules such as PD-L1 nanobody and Coagulase at the tumor site. Overall, this design provides a crucial foundation for constructing a red light–controlled, tumor-targeted therapeutic system with improved precision, efficacy, and biosafety.

![Design Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img22.webp)

Figure 22: Schematic illustration of anchoring module INP-HlpA

## Build

The INP−HlpA-encoding gene was synthesized and codon-optimized for E. coli. Restriction enzyme sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI) were removed to meet the RFC#10 standards and pET28a (m) cloning requirements. The INP-HlpA-encoding gene was cloned into the pET28a (m) vector via the NdeI and XhoI restriction enzyme sites. The recombinant plasmid was transformed into E. coli BL21 by the heat shock method, and positive clones were screened on LB solid plates containing 100 μg/mL kanamycin (Kana) to obtain the recombinant engineered strain BL21−INP−HlpA.

![Build  Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img23.webp)

Figure 23: Construction of the INP-HlpA Expression System and Gene Circuit. (A) Plasmid map. The figure shows the plasmid map of the gene circuit used for INP-HlpA expression. (B) Agarose gel electrophoresis of the INP-HlpA gene fragment. (C) Schematic diagram of the INP-HlpA system gene circuit.

## Test

Induced Expression and Collection of Proteins: The BL21-INP-HlpA strain was cultured overnight in LB broth containing Kana. Subsequently, it was inoculated at a 1% ratio into 30 mL of fresh LB broth and cultured until the OD600 reached 0.2, at which point 0.5 mM IPTG was added to induce expression. The culture temperature was adjusted to 16°C, and cultivation was continued for 20 hours.

Extraction of Intracellular Proteins and WB Verification: 5 mL of the fermentation broth was centrifuged to collect the bacterial cells, which were then resuspended in PBS. Ultrasonic disruption was performed on ice using an ultrasonic disruptor. After centrifugation of the lysate, the supernatant was collected as the intracellular protein sample, and its concentration was determined using the Bradford kit.

Western Blot (WB) Analysis: The intracellular protein samples were separated using a 12.5% SDS-PAGE gel, and transferred to a PVDF membrane via the wet transfer method. After blocking the membrane, it was sequentially incubated with a 1:1000-diluted mouse His−tag antibody (primary antibody) and HRP-labeled goat anti-mouse IgG (secondary antibody). Finally, chemiluminescence analysis was performed using a protein blotting imaging system.

Western Blot analysis results showed that after IPTG induction and low-temperature cultivation for 20 hours, a clear specific band was successfully detected in the intracellular protein sample of the BL21-INP-HlpA strain. The molecular weight of this band was consistent with the expected molecular weight of INP-HlpA and was specifically recognized by the His-tag antibody (approximately 15 - 25 kDa).

![Test Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img24.webp)

Figure 24: WB experimental results of INP-HlpA-his tag

### Adhesion Effect Test

2×10⁵ CT26 cells were inoculated into a 6-well cell culture plate and cultured for 48 hours until the cell confluence reached 80%. Subsequently, the medium was replaced with fresh DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 50 mg/L Amp. Next, 1×10⁷ CFU of the BL21-INP-HlpA-mRFP engineered probiotic was inoculated and co-cultured with CT26 cells for 2 hours. After co-culture, 100 μL of the supernatant was collected, diluted, and spread on Amp-containing plates. The number of free bacteria was quantified by the CFU counting method, and the number of bacteria adhered to the cells and the adhesion efficiency were calculated accordingly. Subsequently, the CT26 cells were washed twice with sterile PBS to remove non-adherent bacteria. Finally, an inverted fluorescence microscope was used to capture mRFP fluorescence images to visually observe the distribution and adhesion morphology of the bacteria on the surface of CT26 cells.

CFU counting results showed that the BL21-INP-HlpA-mRFP strain had a high adhesion efficiency, confirming the specific adhesion effect mediated by HlpA. Images captured by the inverted fluorescence microscope clearly showed that a large number of engineered bacteria labeled with mRFP adhered tightly to the surface of CT26 cells. The results of adhesion rate calculation via CFU counting showed that the adhesion efficiency of the BL21-INP-HlpA-mRFP strain (83.7%) was significantly higher than that of the control group (26.7%). This result confirms the specific adhesion effect mediated by HlpA, and this engineered strain can efficiently target and adhere to CT26 colorectal cancer cells.

![Test Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img25.webp)

Figure 25: Adhesion Effect Test. Figure (1) Adhesion effect diagram of the BL21-INP-HlpA-mRFP engineered strain on CT26 colorectal cancer cells. Figure (2) Comparison of adhesion rates of the engineered strain to CT26 cells (scale bar: 5 μm).

## Learn

Through the successful expression and verification of the INP-HlpA fusion protein, we demonstrated that the designed anchoring module can be efficiently expressed in *E. coli* and maintain stable structural integrity. The clear, specific band detected in the Western Blot confirmed that the INP-HlpA construct was correctly expressed and folded, laying the foundation for its subsequent functional validation.

# Cycle 4-1 MazF

## Design

The MazE/MazF toxin–antitoxin system is a well-characterized bacterial regulatory module that controls growth, dormancy, and programmed cell death. MazF functions as a stable toxin — an mRNA endonuclease that specifically cleaves ACA sequences, thereby blocking protein synthesis and inducing growth arrest or cell death. In contrast, MazE is a labile antitoxin that can bind and neutralize MazF under normal conditions. When MazE is degraded or its synthesis is inhibited, MazF remains active, leading to translational shutdown and cellular dormancy. This system provides a precise, reversible mechanism for controlling bacterial activity.

In our project, the MazE/MazF module is introduced as a biological safety and dormancy control system for the engineered probiotic strain. During the freeze-drying (lyophilization) and storage process, the engineered bacteria enter a dormant state, preserving their viability over long periods. Before use, L-arabinose is supplied externally to induce the expression of MazE, which neutralizes MazF toxicity, ensuring that the bacteria remain alive but inactive. Once administered into the tumor microenvironment, the external arabinose supply is removed, allowing the bacteria to remain in a low-activity, controlled dormant state until activated by red light (NETMAP system). This approach ensures precise temporal control, extended formulation stability, and enhanced biosafety by preventing uncontrolled proliferation while maintaining therapeutic readiness.

![Design Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img26.webp)

Figure 26: Schematic illustration of safety system

To initially construct a MazF toxin safety system based on the lactose-inducible promoter Plac and the LacI repressor protein, and evaluate its ability to control cytotoxicity under non-inducing conditions.

## Build

The MazF toxin gene was synthesized and codon-optimized. The MazF gene was placed downstream of the Plac promoter, and a LacI expression element (PlacI−LacI) was inserted upstream to construct the PlacI−LacI−B0035−Plac−mazF sequence. This sequence was cloned into the pSB1A3 vector and transformed into E. coli DH5α, and an attempt was made to screen positive clones in a medium containing Amp.

![Build  Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img27.webp)

Figure 27: Construction of the Plac−mazF Suicide System. A: Plasmid construction map of the Plac−mazF suicide system. B: Gene circuit diagram of the Plac−mazF suicide system construction.

## Test

No positive clones could be screened out.

## Learn

The Plac promoter has unacceptable low-level leaky expression. Due to the strong toxicity of MazF, even with the inhibitory attempt of the LacI protein, the toxin produced by leakage is sufficient to kill all successfully transformed host cells carrying the plasmid, indicating that the Plac/LacI system is not suitable for the safe control of highly toxic genes.

# Cycle 4-2 PBAD−MazF

## Design

To address the defects of the Plac system, the arabinose-inducible promoter (PBAD) was introduced as a replacement to construct a new MazF safety system, and its tight regulatory ability under non-inducing conditions (i.e., allowing strain survival) was verified. The PBAD promoter is an arabinose-inducible promoter derived from the *araBAD* operon of *Escherichia coli*, and is one of the most tightly regulated and tunable promoters in bacterial genetic engineering. Its expression is controlled by the AraC regulatory protein, which acts as both a transcriptional activator and repressor depending on the presence of L-arabinose.

## Build

The codon-optimized MazF toxin gene was used. The MazF gene was placed downstream of the PBAD promoter to construct the PBAD−mazF sequence. The PBAD−mazF was cloned into the pSB1A3 vector. The recombinant plasmid was transformed into E. coli DH5α by the heat shock method. Positive clones were screened on solid plates containing 100 μg/mL ampicillin (Amp) and verified by sequencing.

## Test

To quantitatively evaluate the function of the PBAD−MazF system under conditions with and without the inducer (arabinose) by monitoring the growth curve, and verify its leak-free safety and induced lethality efficiency.The frozen DH5α−PBAD−MazF strain was activated and inoculated at a ratio of 1:100 into LB medium containing Amp, and the OD600 was adjusted to 0.1. The culture was divided into two groups: the induction group, to which 2% arabinose was added (to induce MazF expression); and the control group, without arabinose addition. At 37°C, a FlexStation 3 was used to continuously monitor the OD600 of the strains in both groups and record the growth curves.

The control group strain showed normal growth. The growth curve of the induction group strain showed severe growth inhibition or complete growth arrest. The normal growth of the control group confirms the high safety (extremely low leakage) of the PBAD system in the non-inducing state.

![Test Diagram](https://static.igem.wiki/teams/5922/engineeringnew/img28.webp)

Figure 28: Growth Curves of the Control Group and Induction Group Strains

## Learn

The growth arrest or inhibition of the induction group confirms the efficient lethality of the MazF toxin, thereby comprehensively verifying that the PBAD−MazF construct is a fully functional and controllable biosafety suicide system.

# Reference

1. Qiao L, Niu L, Wang Z, Deng Z, Di D, Ma X, Zhou Y, Kong D, Wang Q, Yin J, Jin L, Sun J, Feng B, Lu W, Cai F, Guan N, Ye H. Engineered bacteria for near-infrared light-inducible expression of cancer therapeutics. Nat Cancer. 2025 Apr;6(4):612-628. doi: 10.1038/s43018-025-00932-3. Epub 2025 Mar 17. PMID: 40097656.
2. Zhou T, Wu J, Tang H, Liu D, Jeon BH, Jin W, Wang Y, Zheng Y, Khan A, Han H, Li X. Enhancing tumor-specific recognition of programmable synthetic bacterial consortium for precision therapy of colorectal cancer. NPJ Biofilms Microbiomes. 2024 Jan 20;10(1):6. doi: 10.1038/s41522-024-00479-8. PMID: 38245564; PMCID: PMC10799920.
3. Gao T, Niu L, Wu X, Dai D, Zhou Y, Liu M, Wu K, Yu Y, Guan N, Ye H. Sonogenetics-controlled synthetic designer cells for cancer therapy in tumor mouse models. Cell Rep Med. 2024 May 21;5(5):101513. doi: 10.1016/j.xcrm.2024.101513. Epub 2024 Apr 11. PMID: 38608697; PMCID: PMC11148564.
4. Tang, H., Zhou, T., Jin, W., Zong, S., Mamtimin, T., Salama, E. S., ... & Li, X. (2023). Tumor-targeting engineered probiotic Escherichia coli Nissle 1917 inhibits colorectal tumorigenesis and modulates gut microbiota homeostasis in mice. *Life Sciences*, *324*, 121709.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

Content

* [NETMAP module](#section-1)
  + [Construction of a near-infrared (NIR) light–inducible biosensor](#subsection-2)
  + [Testing of the near-infrared (NIR) light–inducible biosensor](#subsection-3)
  + [Effect of irradiation time on the near-infrared (NIR) light–inducible biosensor](#subsection-4)
  + [Flow cytometry analysis](#subsection-5)
  + [Western bloting](#subsection-6)
* [PD-L1 module](#section-7)
  + [Construction of the near-infrared (NIR) light–inducible PD-L1 nanobody (nb) expression system](#subsection-8)
  + [Evaluation of near-infrared (NIR) light–induced PD-L1 nanobody (nb) expression](#subsection-9)
* [Coagulase module](#section-10)
  + [Construction of Coa expression system](#subsection-11)
  + [Coagulase Coagulation Activity](#subsection-12)
* [Adhesion module](#section-13)
  + [Construction of Colorectal Cancer (CRC)–Specific Targeting Strains](#subsection-14)
  + [Evaluation of INP–HlpA Engineered Bacterial Adhesion to CRC Cells](#subsection-15)
* [Suicide module](#section-16)
  + [Construction of the Safety System Based on the lac Promoter](#subsection-17)
  + [Construction of the Safety System Based on the PBAD Promoter](#subsection-18)
  + [Testing of the Safety System](#subsection-19)

# NETMAP module

## Construction of a near-infrared (NIR) light–inducible biosensor

Based on the plasmid maps, the NETMAP NIR light–inducible regulatory module was synthesized (Generalbiol, China). NETMAP comprises two plasmids. Plasmid A is built on the pSB4C5 backbone, carries chloramphenicol resistance, constitutively expresses YhjH and MrkH from the tac promoter, and includes the PmrkA-mRFP cassette. Plasmid B is built on the pSB1A3 backbone, carries ampicillin resistance, and constitutively expresses PadC4 and BphO from the lac promoter. The synthesized sequences were inserted upstream of the mRFP coding region in the pSB1A3 vector using one-step seamless cloning (Seamless Cloning Kit, D7010, Beyotime). The recombinant plasmids were then introduced into E. coli DH5α and BL21 by heat shock (42 °C, 1 min). Positive clones were selected on LB agar plates (1.5% agar) containing ampicillin (100 μg/mL) and chloramphenicol (30 μg/mL) and verified by Sanger sequencing (Tsingke, Beijing), yielding the engineered strains DH5α-NETMAP-mRFP and BL21-NETMAP-mRFP. Strains were stored at −20 °C with 25% (v/v) glycerol as cryoprotectant. Unless otherwise noted, cultures were grown at 37 °C with shaking at 150 rpm, and inoculation/scale-up was performed in LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL ampicillin.

## Testing of the near-infrared (NIR) light–inducible biosensor

To evaluate NIR light–induced gene expression, engineered bacterial strains carrying the NETMAP–mRFP construct were inoculated at a 1:100 ratio into 5 mL of LB medium supplemented with 100 μg/mL ampicillin (Amp) and 30 μg/mL chloramphenicol (Cm). The 15 mL centrifuge tubes were wrapped in aluminum foil to prevent unwanted light exposure, and cultures were incubated at 37 °C with shaking at 180 rpm for 12 hours. After the overnight growth, the bacterial cells were reinoculated at a 1% (v/v) ratio into a 48-well plate containing 500 μL of LB medium supplemented with Amp and Cm, and incubated continuously at 37 °C and 180 rpm until the optical density reached OD₆₀₀ = 0.5. Subsequently, the cultures were irradiated with 660 nm near-infrared (NIR) light using a light-emitting diode (LED) source for 3 hours to induce gene expression.

## Effect of irradiation time on the near-infrared (NIR) light–inducible biosensor

To evaluate the effect of illumination duration on the NETMAP system, samples were exposed to 660 nm NIR light for varying time periods (0–8 hours). A total of 100 μL of bacterial culture was transferred to a 96-well plate, and both OD₆₀₀ and mRFP fluorescence intensity were measured using a microplate reader (FlexStation 3, Molecular Devices, USA) with an excitation wavelength of 584 nm and an emission wavelength of 607 nm. The normalized fluorescence intensity of each sample was calculated as the ratio of fluorescence intensity to OD₆₀₀ (fluorescence/OD₆₀₀).

## Flow cytometry analysis

Engineered bacteria carrying NETMAP–mRFP were cultured until the OD₆₀₀ reached approximately 0.5. One group was maintained under dark conditions, while the other was continuously irradiated with 660 nm NIR light for 3 hours. Following induction, 100 μL of each culture was transferred to a 96-well plate or flow cytometry tube for dilution and sample preparation according to standard flow cytometry protocols. Single-cell fluorescence was detected using a flow cytometer (FlexStation 3, Molecular Devices, USA) to assess fluorescence differences between the dark and light-irradiated groups. Data were collected and analyzed via the PC5.5-A channel (the mRFP fluorescence channel, detecting emission around 607 nm), and the fluorescence intensity distribution of the bacterial population was presented in the form of histograms to visualize population-level expression profiles.

## Western bloting

The coding genes for Coa, YopE1-15 PD-L1 nanobody, and INP-HlpA were synthesized and codon-optimized for E. coli. Restriction enzyme sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI) were removed to comply with RFC#10 standards and pET28a (m) vector cloning requirements. Each gene (Coa, YopE1-15 PD-L1 nb, and INP-HlpA) was inserted into the pET28a (m) vector via NdeI and XhoI restriction sites. The recombinant plasmids were then transformed into competent E. coli BL21 cells using the heat shock method (42°C, 1 min). Positive transformants were selected on LB agar plates (supplemented with 1.5% agar) containing 100 μg/mL kanamycin (Kana) and confirmed by Sanger sequencing (Tsingke, Beijing). The recombinant strains BL21–Coa, BL21–YopE1-15 PD-L1 nb, and BL21–INP-HlpA were successfully obtained. Strains were stored at –20°C in 25% (v/v) glycerol as a cryoprotectant. For cultivation, engineered strains were grown in LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL kanamycin at 37°C and 150 rpm.

For protein induction, frozen bacterial cultures stored at –80°C were inoculated at a 1:100 ratio into 5 mL of LB broth (supplemented with 100 μg/mL kanamycin) in a 15 mL centrifuge tube and cultured overnight at 37°C with shaking at 150 rpm. Then, 1% (v/v) of the culture was re-inoculated into 30 mL of fresh LB broth (A507002, Sangon Biotech) and cultured at 37°C and 180 rpm for 1–2 hours. When the OD₆₀₀ reached 0.2, 0.5 mM IPTG was added to induce protein expression. The culture temperature was adjusted to 16°C, and incubation continued for 20 hours to enhance soluble protein production.

After fermentation, 5 mL of culture was collected by centrifugation (10,000 × g, 5 min). The bacterial pellet was resuspended in PBS, and ultrasonic disruption was performed on ice using an ultrasonic disruptor (Shanghai Jinxin, China) under the following conditions: 1 s on / 3 s off cycle, 70 W power, total 20 min. The lysate was centrifuged at 10,000 × g, 4°C for 30 min (Biosafer1000, Saifei), and the supernatant was collected as the intracellular protein sample. Protein concentration was determined using the Bradford Protein Assay Kit (P0006, Beyotime).

Protein samples were separated using a 12.5% SDS-PAGE gel prepared with the One-step PAGE Gel Rapid Preparation Kit (PG113, Yeasen). The separated proteins were transferred onto a 0.22 μm PVDF membrane (WJ001S, Yeasen) via wet transfer. The membrane was blocked at room temperature for 20 minutes with a protein-free rapid blocking buffer (ED0024, Sikejie), followed by incubation with a 1:1000-diluted mouse anti-His-tag antibody (AH367, Beyotime) as the primary antibody at 4°C overnight. After washing, the membrane was incubated with an HRP-labeled goat anti-mouse IgG (H+L) secondary antibody (A0216, Beyotime) at room temperature for 1 hour. Finally, chemiluminescence detection was performed using a protein blot imaging system (ChemiDoc MP, Bio-Rad) to visualize the target protein bands.

# PD-L1 module

## Construction of the near-infrared (NIR) light–inducible PD-L1 nanobody (nb) expression system

The YopE1-15 PD-L1 nanobody (PD-L1 nb) gene (Generalbiol, China) was synthesized and codon-optimized for E. coli, with the EcoRI, XbaI, SpeI, and PstI restriction enzyme sites removed to comply with RFC#10 assembly standards. The mRFP reporter gene in the previously constructed NIR light–inducible biosensor (NETMAP) was replaced with the YopE1-15 PD-L1 nb sequence using the one-step seamless cloning method (Seamless Cloning Kit, D7010, Beyotime). The resulting recombinant plasmid was transformed into E. coli DH5α using the heat shock method (42°C, 1 min). Positive clones were screened on LB agar plates (supplemented with 1.5% agar) containing 100 μg/mL ampicillin (Amp) and verified by DNA sequencing (Tsingke, Beijing). The successfully engineered recombinant strain, DH5α–NETMAP–PD-L1 nb, was obtained and stored at –20°C in 25% (v/v) glycerol as a cryoprotectant. The engineered strain was cultured at 37°C with shaking at 150 rpm, and for inoculation and scale-up, LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL Amp was used as the growth medium.

## Evaluation of near-infrared (NIR) light–induced PD-L1 nanobody (nb) expression

The DH5α–NETMAP–PD-L1 nb strain was inoculated at a 1:100 ratio into 5 mL of LB medium containing 100 μg/mL ampicillin (Amp). The 15 mL centrifuge tubes were wrapped with aluminum foil to prevent light exposure. The culture was grown at 37°C and 180 rpm for 12 hours. Subsequently, the bacterial cells were inoculated at a 1% rate into 48-well plates containing 500 μL of Amp⁺ LB medium and further cultured at 37°C and 180 rpm until the OD₆₀₀ reached 0.5. At this point, samples were irradiated with 660 nm near-infrared light (NIR) using a light-emitting diode (LED) setup for 7 hours. After induction, the supernatant of the NETMAP-engineered bacterial culture was collected and centrifuged at 12,000 × g for 10 minutes at 4°C. The supernatant was then filtered through a 0.22 μm filter membrane, followed by concentration using a BeyoGold™ ultrafiltration tube (15 mL, 5 kDa MWCO, PES; FUF505, Beyotime) by centrifugation at 4,000 × g for 40 minutes at 4°C. The protein concentration of the concentrated sample was determined using the Bradford Assay Kit (P0006, Beyotime). For Western blot analysis, the concentrated protein samples were separated using a 12.5% SDS-PAGE gel prepared with the One-step PAGE Gel Rapid Preparation Kit (PG113, Yeasen). The proteins were then transferred onto a 0.22 μm PVDF membrane (WJ001S, Yeasen) via wet transfer. The membrane was blocked with a protein-free rapid blocking buffer (ED0024, Sikejie) at room temperature for 20 minutes, followed by incubation with a 1:1000-diluted mouse anti-His-tag antibody (AH367, Beyotime) as the primary antibody at 4°C overnight. After washing, the membrane was incubated with an HRP-labeled goat anti-mouse IgG (H+L) secondary antibody (A0216, Beyotime) at room temperature for 1 hour.

# Coagulase module

## Construction of Coa expression system

For the preparation of recombinant strains, induced expression, and crude protein extraction: After codon optimization of the Coagulase-encoding gene and removal of restriction enzyme sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI), the gene was cloned into the pET28a (m) vector via the NdeI and XhoI restriction enzyme sites. The recombinant plasmid was transformed into competent E. coli BL21 strain by the heat shock method (42°C, 1 min). Transformants were screened on LB solid plates containing 100 μg/mL kanamycin (Kana) to obtain the recombinant engineered strain BL21-Coa. After overnight culture of the BL21-Coa strain in LB broth containing Kana, it was inoculated at a ratio of 1:100 into 10 mL of fresh medium and cultured at 37°C with shaking at 180 rpm until the OD600 reached 0.5-0.8.

## Coagulase Coagulation Activity

Non-anticoagulated blood samples were used. Subsequently, 50 μL of purified Coa protein solutions with different concentrations were mixed with 50 μL of blood samples in EP tubes or 96-well plates, with a total reaction volume of 100 μL. The concentration gradient of the Coa protein solution included 100%, 80%, 60%, 40%, and 20%, and a mixture of 0% Coa solution (i.e., buffer) and 50 μL of blood sample was used as the negative control. Meanwhile, 100 μL of water and 100 μL of 100% Coa solution were set as blank controls without blood. All mixtures were incubated in a 37°C incubator, and photos were taken every 5 minutes to record the coagulation status for 30 minutes. A final photo was taken at 60 minutes when complete coagulation was achieved. The main observation and analysis indicators included thrombosis formation time, thrombus morphology, and coagulation intensity.

# Adhesion module

## Construction of Colorectal Cancer (CRC)–Specific Targeting Strains

A truncated ice nucleation protein (INP) was fused to the N-terminus of HlpA to display the HlpA protein on the surface of engineered probiotics. Additionally, mRFP was fused downstream of HlpA to enable visual observation of HlpA-mediated adhesion to colorectal cancer cells. The INP–HlpA–mRFP sequence (Generalbiol, China) was synthesized and codon-optimized for E. coli, with the EcoRI, XbaI, SpeI, and PstI restriction sites removed to comply with RFC#10 assembly standards. The J23100-B0034 cis-regulatory elements (constitutive promoter and RBS) were introduced upstream of the INP–HlpA sequence to drive continuous expression. The fusion gene was cloned into the pSB1A3 vector via the XbaI and SpeI restriction enzyme sites. The recombinant plasmid was transformed into E. coli BL21 using the heat shock method (42°C, 1 min). Positive clones were selected on LB agar plates (supplemented with 1.5% agar) containing 100 μg/mL ampicillin (Amp) and verified by Sanger sequencing (Tsingke, Beijing). The resulting recombinant strain, BL21–INP–HlpA, was obtained and stored at –20°C in 25% (v/v) glycerol as a cryoprotectant. The engineered strain was cultured at 37°C with shaking at 150 rpm, and inoculation and scale-up were performed in LB broth (G3102, Servicebio, China) supplemented with 100 μg/mL Amp.

## Evaluation of INP–HlpA Engineered Bacterial Adhesion to CRC Cells

To assess the adhesion ability of the engineered strain, CT26 colorectal cancer cells were seeded at a density of 2 × 10⁵ cells per well in a 6-well culture plate and incubated for 48 hours until reaching 80% confluence. The culture medium was replaced with fresh DMEM supplemented with 50 mg/L Amp. Subsequently, 1 × 10⁷ CFU of engineered probiotics were added and co-cultured with CT26 cells for 2 hours. After co-incubation, 100 μL of the supernatant was collected, serially diluted, and spread onto Amp-containing LB plates to determine the number of non-adherent (free) bacteria by CFU counting, allowing calculation of adhesion efficiency. The CT26 cells were washed twice with sterile PBS to remove unbound bacteria, and fluorescence images of adherent bacteria expressing mRFP were captured using an inverted fluorescence microscope to visualize bacterial adhesion on the cell surface.

# Suicide module

## Construction of the Safety System Based on the lac Promoter

As previously described, the E. coli toxin protein MazF was synthesized and codon-optimized to comply with RFC#10 standards. The MazF gene was placed downstream of the lactose-inducible promoter Plac, generating the construct Plac–mazF. Additionally, a LacI expression cassette (PlacI–LacI) was inserted upstream of Plac–mazF and separated by the transcriptional terminator B0015, resulting in the complete regulatory sequence PlacI–LacI–B0035–Plac–mazF. The assembled construct was cloned into the pSB1A3 vector via the XbaI and SpeI restriction sites, and the recombinant plasmid was transformed into E. coli DH5α by the heat shock method (42°C, 1 min).

## Construction of the Safety System Based on the PBAD Promoter

Similarly, the E. coli toxin protein MazF was synthesized and codon-optimized according to RFC#10 standards. The MazF gene was placed under the control of the arabinose-inducible promoter PBAD, forming the construct PBAD–mazF. This sequence was cloned into the pSB1A3 vector via the XbaI and SpeI restriction sites and transformed into E. coli DH5α using the heat shock method (42°C, 1 min). Positive transformants were screened on LB agar plates (supplemented with 1.5% agar) containing 100 μg/mL ampicillin (Amp) and verified by DNA sequencing (Tsingke, Beijing). The successfully constructed recombinant strain, DH5α–PBAD–MazF, was stored at –20°C in 25% (v/v) glycerol as a cryoprotectant. The strain was cultured at 37°C with shaking at 150 rpm, and inoculation and expansion were performed in LB broth (G3102, Servicebio, China) containing 100 μg/mL Amp.

## Testing of the Safety System

The frozen DH5α–PBAD–MazF strain stored at –20°C was revived by inoculating into 5 mL of LB medium containing Amp and cultured overnight at 37°C and 150 rpm. The overnight culture was then inoculated at a 1:100 ratio into 20 mL of fresh LB medium with Amp, and the OD₆₀₀ was adjusted to 0.1. One group was supplemented with 2% L-arabinose (A83229, Innochem) to induce MazF expression, while the control group was grown without arabinose. The growth curves of both groups were continuously monitored using a FlexStation 3 microplate reader (Molecular Devices, USA) to evaluate the effect of MazF induction on bacterial viability.

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Content

* [Abstract](#section-1)
  + [System 1: Red Light-Induced System](#section-2)
    - [Construction of NIR Light-Induced Biosensor](#subsection-3)
    - [Response of the NETMAP Promoter to 660 nm NIR Light](#subsection-4)
    - [Flow Cytometric Analysis of Single-Cell Fluorescence Distribution](#subsection-5)
    - [Effect of Induction Time on the Dynamic Response of the NETMAP System](#subsection-6)
    - [Effect of Chassis Strains on the Performance of the NETMAP NIR Light-Induced System](#subsection-7)
    - [PD-L1 nb Protein Expression and Western Blot Verification](#subsection-8)
    - [Construction of the NIR Light-Induced YopE1-15-PD-L1 Nanobody Expression System](#subsection-9)
    - [Verification of NIR Light-Induced Expression and Extracellular Secretion of YopE1-15-PD-L1 Nanobody](#subsection-10)
    - [System 2: Therapeutic System - Expression, Purification, and Activity Verification of Coagulase (Coa)](#subsection-11)
    - [Expression, Purification, and WB Verification of Coagulase Protein](#subsection-12)
    - [Verification of Coagulase Coagulation Activity](#subsection-13)
  + [System 3: Construction and Functional Verification of the Targeting System](#section-14)
    - [Expression and Western Blot Verification of the INP-HlpA Targeting Protein](#subsection-15)
    - [Construction of the Targeting Strain](#subsection-16)
    - [Adhesion Effect Test of the INP-HlpA Engineered Strain on Colorectal Cancer CT26 Cells](#subsection-17)
  + [System 4: Construction and Evaluation of the Biosafety Suicide System](#section-18)
    - [Construction of the Suicide System Based on the Plac Promoter](#subsection-19)
    - [Construction of the Suicide System Based on the PBAD Promoter](#subsection-20)
    - [Functional Test of the PBAD−MazF Suicide System](#subsection-21)

# Abstract

This study aimed to construct a multifunctional and controllable engineered probiotic platform for targeted tumor therapy. This platform integrates four core systems: a red light-induced switch, tumor-targeting capability, therapeutic payload expression, and a biosafety suicide mechanism. The red light-induced system (NETMAP) was successfully constructed, achieving precise response to 660 nm near-infrared (NIR) light via the PmrkA promoter. Functional verification demonstrated that the system exhibits extremely low biological noise and high uniformity in activation (validated by flow cytometry), with the optimal induction time determined to be 5 hours. For the chassis strain selection, DH5α was identified as the optimal host due to its extremely low background leakage (FC ≈ 4.4). Finally, this light-controlled switch was successfully applied to drive the expression and efficient extracellular secretion of the YopE1−15-fused PD−L1 nanobody (nb), confirming the practicality of the system. The targeting system, by displaying the HlpA adhesion protein on the surface of E. coli, achieved efficient and specific adhesion to CT26 colorectal cancer cells, laying a foundation for the enrichment of engineered bacteria at tumor sites. The core therapeutic payload, Coagulase (Coa) protein, was successfully expressed, purified, and its concentration-dependent coagulation activity was verified through in vitro coagulation assays. Eventually, the construction of the biosafety suicide system addressed the issue of leaky expression of the highly toxic protein MazF, leading to the successful development of the PBAD−MazF system. This system demonstrates high safety (normal growth) in the absence of an inducer and can efficiently induce suicide when induced, ensuring precise control of engineered bacteria and environmental safety. The successful construction and verification of these systems collectively provide a comprehensive technical foundation for the development of controllable, safe, and efficient engineered bacteria for targeted tumor therapy.

# System 1: Red Light-Induced System

In this study, a gene expression regulatory system (NETMAP) based on NIR light induction was successfully constructed to achieve remote and precise control of biological functional modules. The PmrkA promoter was utilized to respond to 660 nm NIR light signals. During the system construction and verification phase, functional tests were first conducted in DH5α and BL21 strains using mRFP as the reporter gene. It was confirmed that the NETMAP promoter could be significantly activated under 660 nm light irradiation, with the normalized fluorescence intensity being significantly higher than that of the dark control group. Single-cell flow cytometry analysis further confirmed the excellent performance of the system, as light irradiation enabled the cell population to uniformly transition from a low-expression "off" state to a high-expression "on" state. Dynamic response experiments determined that the optimal induction time of the system was 5 hours, at which point the mRFP expression reached its peak, indicating that the system possesses time-dependent controllable characteristics. Chassis strain comparison experiments concluded that the DH5α strain, due to its extremely low background leakage and higher induction fold (4.5-fold), was selected as the optimal chassis strain. Finally, the mRFP gene was replaced with the sequence of the YopE1−15-fused PD−L1 nanobody (nb) for tumor immunotherapy, successfully constructing the DH5α−NETMAP−PDL1 nb expression system. Subsequent Western Blot analysis verified that under 660 nm NIR light induction, this system could successfully drive the expression of the target nanobody, and the YopE1−15 sequence mediated the efficient extracellular secretion of the PD−L1 nb protein. This confirms the practicality and functionality of the NETMAP system as a remote light-controlled switch for immunotherapy.

## Construction of NIR Light-Induced Biosensor

### Objective

To construct a gene expression regulatory system based on NIR light induction, serving as the remote-controlled switch module in our study. We selected the NETMAP promoter as the light-responsive element and used the red fluorescent protein mRFP as the reporter gene to verify the system's functionality.

### Methods

The NIR light-induced promoter NETMAP was synthesized by gene synthesis (Generalbiol, China). The NETMAP system consists of two plasmids. Plasmid A, based on the pSB4C5 backbone, contains chloramphenicol resistance, constitutively expresses YhjH and MrkH using the tac promoter, and carries PmrkA-mRFP. Plasmid B, based on the pSB1A3 backbone, contains ampicillin resistance and constitutively expresses PadC4 and BphO using the lac promoter. The above-synthesized sequences were cloned upstream of the mRFP coding sequence in the pSB1A3 vector via the one-step cloning method (Seamless Cloning Kit, D7010, beyotime). Subsequently, the recombinant plasmids were transformed into E. coli DH5α and BL21 by the heat shock method (42°C, 1 min). Positive clones were screened on LB (Luria Bertani) solid plates (supplemented with 1.5% agar) containing 100 μg/mL ampicillin (Amp) and 30 μg/mL chloramphenicol (Cm), and verified by sequencing (Tsingke, Beijing), resulting in the acquisition of recombinant engineered strains DH5α-NETMAP-mRFP and BL21-NETMAP-mRFP. The strains were stored at -20°C with 25% (v/v) glycerol used as an antifreeze agent. The engineered strains were cultured at 37°C with shaking at 150 rpm. Inoculation and expansion culture were performed in LB broth (G3102, Servicebio, China) containing 100 μg/mL Amp.

### Results

The pSB4C5-YhjH-MrkH plasmid, pSB1A3-PadC4-BphO plasmid, and pSB1A3-PadC4-BphO-YhjH-MrkH plasmid were successfully constructed.

![Construction of NIR Light-Induced Biosensor Diagram](https://static.igem.wiki/teams/5922/results/img1.webp)

Figure 1: Construction of NETMAP (A1) The plasmid map of pSB4C5-YhjH-MrkH. (A2) The plasmid map of pSB1A3-PadC4-BphO. (A3) The plasmid map of NETMAP. (B) Agarose gel electrophoresis of BphO (591 bp), PadC4 (2037 bp), MrkH (705 bp), and YhjH (768 bp). (C) The gene circuit of NETMAP.

## Response of the NETMAP Promoter to 660 nm NIR Light

### Objective

This experiment aimed to quantitatively evaluate the specific response efficiency of the NETMAP promoter to 660 nm NIR light and determine the basal expression level of the system in the absence of light, thereby confirming the function of NETMAP as a light-induced gene switch. Meanwhile, this verification also included the optimization of the induction environment to ensure that the obtained functional verification data are based on stable and reproducible experimental conditions. Methodological factors such as stray light interference, temperature fluctuations, and light obstruction were excluded to avoid affecting the evaluation of system performance, thus improving the reliability of the experimental results.

### Methods

The experiment began with the optimization of the light irradiation environment (as shown in Figures A and B). Initially, an attempt was made to use an LED for light irradiation inside a clean bench (Figure A). However, due to stray light interference in the environment, obstruction of light by the test tube rack, and difficulty in maintaining a constant temperature of 37°C, the initial induction effect was unsatisfactory. Therefore, an optimized method was adopted: the LED light source was fixed inside a closed incubator (Figure B) to ensure complete isolation from external stray light. The uniformity and utilization efficiency of light were enhanced through the reflection of the stainless-steel inner wall, while the culture temperature was precisely controlled at 37°C, ensuring that all subsequent functional verification experiments were conducted under optimal and stable conditions. After establishing a stable environment, functional verification was initiated: the engineered strain carrying the NETMAP-mRFP gene circuit (BL21-NETMAP-mRFP) was inoculated at a ratio of 1:100 into 5 mL of LB liquid medium containing 100 μg/mL Amp, and activated and cultured overnight (12 hours) in a shaker at 37°C and 180 rpm. To ensure the accuracy of subsequent experimental controls, all culture tubes were wrapped with tin foil to achieve complete light shielding. On the next day, the activated bacterial culture was transferred at a 1% inoculation rate into 500 μL of fresh Amp⁺ LB medium in a 48-well plate, followed by continued culture at 37°C and 180 rpm until the bacterial density reached an OD600 of approximately 0.5. Once the predetermined OD value was achieved, the samples in the experimental group were transferred to a closed incubator and irradiated with a 660 nm NIR light LED for 3 hours. The samples in the dark control group were continuously cultured under complete light-shielded conditions. After the induction period, bacterial samples from both groups were collected simultaneously. A microplate reader was used to measure the OD600 and mRFP fluorescence intensity (excitation wavelength: 584 nm, emission wavelength: 607 nm) of the bacteria synchronously. Subsequently, the normalized fluorescence intensity (fluorescence intensity divided by OD600) was calculated to eliminate potential differences in bacterial density among different samples, ensuring accurate and fair quantitative comparison of the actual induction efficiency of the NETMAP promoter.

### Results

Results after optimizing the experimental environment (as shown in Figure B) indicated that the normalized mRFP fluorescence intensity of the bacterial strain in the experimental group irradiated with 660 nm NIR light for 3 hours was significantly higher than that of the dark control group cultured under complete light shielding throughout the experiment (as shown in Figure C). This result demonstrates that 660 nm NIR light can be effectively perceived and responded to as a signal by the NETMAP promoter, and the NETMAP promoter was successfully activated. Meanwhile, as shown in Figure D, within the 3-hour experimental period, there was no statistically significant difference in the OD600 values between the light-irradiated group and the dark control group, indicating that 660 nm NIR light had no significant impact on the bacterial growth rate and ruling out the possibility of inhibitory toxicity of NIR light on bacterial growth.

![Response of the NETMAP Promoter to 660 nm NIR Light Diagram](https://static.igem.wiki/teams/5922/results/img2.webp)

Figure 2: Experimental Optimization and Functional Verification of the NIR Light-Induced System. (A) Schematic diagram of light irradiation for the NETMAP system in the initial clean bench environment. (B) Optimized light irradiation environment for the NETMAP system in a closed incubator. (C) Comparison of normalized fluorescence intensity of the engineered strain after 3 hours of induction in the dark and under 660 nm NIR light. (D) Growth curves of the engineered strain under 660 nm NIR light and dark conditions.

### Conclusion

The NETMAP promoter functions effectively under 660 nm NIR light induction, as evidenced by the significantly higher normalized mRFP fluorescence intensity in the light-irradiated group compared to the dark control group. This result confirms the suitability of the 660 nm wavelength and verifies the basic function of the NETMAP system as a light-controlled switch.

## Flow Cytometric Analysis of Single-Cell Fluorescence Distribution

### Objective

This experiment aimed to accurately evaluate the activation uniformity and induction quality of the NETMAP gene switch at the single-cell resolution. By comparing the fluorescence distribution curves of the dark control and light-induced groups, it was determined whether the NETMAP system could enable the cell population to uniformly transition to a high-expression state when activated, thereby supplementing and verifying the population-averaged data obtained by the microplate reader.

### Methods

The culture and induction conditions of the strains were consistent with those in the macroscopic fluorescence analysis experiment: the engineered strain carrying NETMAP-mRFP was cultured until the OD600 reached approximately 0.5, after which one group was placed in the dark and the other was continuously irradiated with 660 nm NIR light for 3 hours. Referring to standard flow cytometry techniques, after the induction, 100 μL of the culture was taken and transferred to a 96-well plate or flow tube for dilution and sample preparation. Subsequently, a flow cytometer (FlexStation 3, Molecular Devices, USA) was used for single-cell fluorescence detection to assess the fluorescence changes of the engineered strain under dark conditions and 660 nm light excitation. Data were collected and analyzed via the PC5.5-A channel (the mRFP fluorescence channel, corresponding to signals near the emission wavelength of 607 nm), and the fluorescence distribution characteristics of the cell population were ultimately presented in the form of a fluorescence intensity distribution histogram.

### Results

The flow cytometric analysis chart (as shown in the figure) clearly illustrates the fluorescence intensity distribution of the cell population. The fluorescence distribution of the dark control group (gray peak) was concentrated in the region of low fluorescence intensity, indicating an extremely low basal expression level of mRFP in the absence of light induction. In contrast, the fluorescence distribution of the 660 nm NIR light-induced group (red peak) showed a significant and uniform shift toward the right (high fluorescence intensity). Additionally, both peak shapes remained relatively concentrated with a single-peak structure, and no obvious double-peak or scattered phenomenon was observed after light induction.

![Flow Cytometric Analysis of Single-Cell Fluorescence Distribution Diagram](https://static.igem.wiki/teams/5922/results/img3.webp)

Figure 3: Flow Cytometric Analysis of Fluorescence Distribution of Engineered Strains in the Dark and Under NIR Light.

### Conclusion

Flow cytometric analysis confirmed the excellent quality of the NETMAP light-controlled gene switch. 660 nm NIR light not only increased the average expression level of mRFP but also achieved efficient and uniform induction of the cell population. Light irradiation enabled the entire cell population to uniformly transition from a low-expression "off" state to a high-expression "on" state, indicating that the NETMAP system exhibits good induction stability and low biological noise at the single-cell level.

## Effect of Induction Time on the Dynamic Response of the NETMAP System

### Objective

This experiment aimed to quantitatively determine the dynamic response characteristics of the NETMAP promoter system under continuous induction with 660 nm NIR light, i.e., to study the expression accumulation rate and maximum expression level of the mRFP reporter gene, thereby identifying the optimal induction time of the system.

### Methods

The engineered strain carrying the NETMAP-mRFP gene was cultured until the OD600 reached approximately 0.5. One group was placed in a closed incubator and irradiated with a 660 nm LED NIR light, while the other group served as the dark control. A light irradiation time gradient ranging from 0 to 8 hours was set. At predetermined time points (e.g., 1 h, 3 h, 5 h, 8 h), samples were collected simultaneously from the light-irradiated group and the dark control group. After sampling, a microplate reader was used to measure the OD600 and mRFP fluorescence intensity of the bacteria synchronously. All data were calculated as normalized fluorescence intensity to analyze the change curve of mRFP expression over time. The maximum induction time was set to 8 hours, considering the avoidance of fluorescence quenching of the reporter protein mRFP under prolonged red light irradiation.

### Results

The experimental results (as shown in the figure) clearly demonstrate the dynamic response process of the NETMAP system. The normalized fluorescence intensity of the dark control group remained at an extremely low level within 8 hours, reconfirming the excellent low-background characteristic of the system. The normalized fluorescence intensity of the 660 nm light-irradiated group accumulated rapidly with the extension of time, and the expression level increased continuously within the initial 0 to 5 hours. The curve reached its peak at 5 hours, with a normalized fluorescence intensity of approximately 32 A.U. After 5 hours, the expression level slightly decreased, which may be attributed to the degradation of the reporter protein, fluorescence quenching, or increased cellular metabolic stress.

![Effect of Induction Time on the Dynamic Response of the NETMAP System Diagram](https://static.igem.wiki/teams/5922/results/img4.webp)

Figure 4: Relationship Between Induction Time and Fluorescence Intensity of the NETMAP Engineered Strain

### Conclusion

The NETMAP system exhibits a clear time-dependent response to 660 nm NIR light, and its induction process is controllable. The expression level of the mRFP reporter protein accumulates with the extension of light irradiation time, and the optimal induction time point of the system is determined to be 5 hours, at which the maximum normalized fluorescence output can be obtained. This result provides key parameter support for the precise control of the expression level and duration of target genes in practical applications.

## Effect of Chassis Strains on the Performance of the NETMAP NIR Light-Induced System

### Objective

This experiment aimed to compare the response differences of two commonly used E. coli chassis strains (DH5α and BL21) to the NETMAP NIR light-induced system. By quantifying the absolute fluorescence output intensity and induction fold (FC) in different chassis strains, data support was provided for the subsequent selection of the most suitable engineered strain.

### Methods

The recombinant plasmid carrying the NETMAP-mRFP gene was transformed into E. coli DH5α and BL21 strains, respectively. Both engineered strains were induced when the OD600 reached approximately 0.5 and simultaneously irradiated with 660 nm NIR light for 3 hours in a closed, constant-temperature environment, with respective dark control groups set up. After induction, a microplate reader was used to measure the OD600 and mRFP fluorescence intensity of all samples. Data processing included: calculating the normalized fluorescence intensity of each sample to evaluate the absolute expression intensity; and calculating the induction fold (normalized fluorescence intensity of the light-irradiated group / normalized fluorescence intensity of the dark control group) of each strain to evaluate the switch sensitivity.

### Results

When comparing the responses of the two chassis strains to the NETMAP system, two key parameters were analyzed: absolute output intensity and induction fold (FC). As shown in Figure A, after 3 hours of induction with 660 nm light irradiation, the normalized fluorescence intensity of the BL21 strain in the light-irradiated group (approximately 24.5 A.U.) was slightly higher than that of the DH5α strain (approximately 16.5 A.U.), indicating that BL21 has a slight advantage in the absolute expression yield of the gene. However, this advantage is accompanied by a significant drawback: under dark conditions, the basal background leakage of the BL21 strain (approximately 13.5 A.U.) is much higher than that of the DH5α strain (approximately 3.7 A.U.). This high background leakage directly affects the "switch" quality of the system. As shown in the comparison of induction folds in Figure B, the induction fold of the DH5α strain (approximately 4.5-fold) is significantly higher than that of the BL21 strain (approximately 1.8-fold). This difference is a direct result of the extremely low background leakage of the DH5α strain, leading to a higher ratio of light-irradiated to dark conditions, thereby demonstrating stronger switch sensitivity and a better signal-to-noise ratio.

![Effect of Chassis Strains on the Performance of the NETMAP NIR Light-Induced System Diagram](https://static.igem.wiki/teams/5922/results/img5.webp)

Figure 5: Effect of Chassis Strains on the Performance of the NETMAP System. (A) Comparison of normalized fluorescence intensity of different chassis strains in the dark and under 660 nm NIR light. (B) Comparison of fluorescence induction folds (FC) of different chassis strains.

### Conclusion

The selection of chassis strains has a significant impact on the performance of the NETMAP system. Considering that the purpose of this system is to serve as a precise switch, the DH5α strain is a more suitable choice for the chassis strain due to its stronger switch sensitivity (high FC value) and lower basal leakage.

## PD-L1 nb Protein Expression and Western Blot Verification

### Objective

This experiment aimed to achieve and verify the efficient expression of the PD-L1 nanobody (nb)-encoding gene in the E. coli BL21 strain via the pET28a induction system.

### Methods

Construction of Recombinant Plasmids and Preparation of Engineered Strains: First, codon optimization was performed on the PD-L1 nb-encoding gene for E. coli, and restriction enzyme sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI) were removed to meet the RFC#10 standards and pET28a (m) cloning requirements. Subsequently, the target gene was cloned into the pET28a (m) vector via the NdeI and XhoI restriction enzyme sites. The recombinant plasmid was transformed into competent E. coli BL21 strain by the heat shock method (42°C, 1 min). Transformants were screened on LB solid plates containing 100 μg/mL kanamycin (Kana). After verification of positive clones by sequencing, the recombinant engineered strain BL21-PD-L1 nb was obtained and stored in a culture medium containing 25% (v/v) glycerol (-20°C).

Induced Expression and Collection of Proteins: The bacterial culture frozen at -80°C was inoculated at a ratio of 1:100 into a centrifuge tube containing 5 mL of LB broth (supplemented with 100 μg/mL Kana) and cultured overnight at 37°C with shaking at 150 rpm. Subsequently, 1% (v/v) of the culture was re-inoculated into 30 mL of fresh LB broth and continuously cultured at 37°C with shaking at 180 rpm for 1-2 hours. When the OD600 reached 0.2, 0.5 mM IPTG was added to the medium to induce the expression of the target protein. The culture temperature was then adjusted to 16°C, and cultivation was continued for 20 hours to promote the formation of soluble proteins.

Extraction and Quantification of Intracellular Proteins: 5 mL of the fermentation broth was centrifuged (10,000×g, 5 min), and the bacterial pellet was resuspended in PBS. Ultrasonic disruption was performed on ice using an ultrasonic disruptor (ultrasonication for 1 s, interval of 3 s, 70 W, 20 min). The sonicated lysate was centrifuged at 10,000×g for 30 min at 4°C, and the supernatant was collected as the intracellular protein sample. The protein concentration was determined using the Bradford kit (P0006).

Western Blot (WB) Verification: The intracellular protein samples were separated using a 12.5% SDS-PAGE gel prepared with the one-step PAGE gel rapid preparation kit (PG113). The separated proteins were transferred to a 0.22 μm PVDF membrane via the wet transfer method. The membrane was blocked with protein-free rapid blocking solution (ED0024) at room temperature for 20 min, followed by incubation with a 1:1000-diluted mouse His-tag antibody (AH367) (primary antibody) at 4°C overnight. Finally, the membrane was incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (H+L) (A0216) (secondary antibody) at room temperature for 1 hour. Chemiluminescence analysis was performed using a protein blotting imaging system.

![PD-L1 nb Protein Expression and Western Blot Verification Diagram](https://static.igem.wiki/teams/5922/results/img6.webp)

Figure 6: Construction of the PD-L1 nb System and Gene Circuit. (A) Plasmid map. The figure shows the plasmid map of the gene circuit used for PD-L1 nb expression. (B) Schematic diagram of the gene circuit of the PD-L1 nb system.

### Results

Western Blot analysis results showed that after IPTG induction and low-temperature cultivation for 20 hours, a clear specific band was successfully detected in the intracellular protein sample of the BL21-PD-L1 nb strain. The molecular weight of this band was consistent with the expected molecular weight of PD-L1 nb (between approximately 15-20 kDa) and was specifically recognized by the His-tag antibody. This confirms the successful expression of the target gene.

![PD-L1 nb Protein Expression and Western Blot Verification Diagram](https://static.igem.wiki/teams/5922/results/img7.webp)

Figure 7: WB Analysis of Bacterial Lysates from E. coli BL21 Carrying the pET28a Recombinant Vector After Induction with 0.5 mM IPTG at 16°C for 20 Hours

### Conclusion

The PD-L1 nb-encoding gene achieved efficient induced expression in the BL21 strain via the pET28a system.

## Construction of the NIR Light-Induced YopE1-15-PD-L1 Nanobody Expression System

### Objective

This experiment aimed to combine the light-controlled switch module (NETMAP promoter) with functional therapeutic elements by replacing the reporter gene mRFP, thereby constructing a PD-L1 nanobody (nb) expression system that can be precisely regulated by NIR light. The target product is YopE1-15-fused PD-L1 nb, which is used to achieve tumor immunotherapy functions.

### Methods

First, the sequence encoding YopE1-15-fused PD-L1 nb was synthesized by gene synthesis (Generalbiol, China). This sequence was codon-optimized for E. coli, and common BioBrick restriction enzyme sites (EcoR I, Xba I, Spe I, and Pst I) were removed to ensure compliance with RFC#10 standards and facilitate subsequent cloning. Subsequently, the one-step cloning method (Seamless Cloning Kit, D7010, beyotime) was used to replace the mRFP reporter gene in the previously constructed NIR light-induced biosensor with this YopE1-15-PDL1 nb sequence, thereby placing the expression of PDL1 nb under the control of the NETMAP promoter. The obtained recombinant plasmid was transformed into the preferred chassis strain E. coli DH5α by the heat shock method (42°C, 1 min). Transformants were screened on LB solid plates containing 100 μg/mL ampicillin (Amp) and 30 μg/mL chloramphenicol (Cm). Positive clones were finally confirmed by sequencing verification (Tsingke). The obtained DH5α-NETMAP-PDL1 nb engineered strain was stored at -20°C with 25% (v/v) glycerol and inoculated and expanded at 37°C with shaking at 150 rpm.

### Results

Through gene synthesis, codon optimization, and one-step cloning technology, the recombinant plasmid driven by the NETMAP promoter for the expression of YopE1-15-PD-L1 nb was successfully constructed. After antibiotic screening and sequencing verification, the DH5α-NETMAP-PDL1 nb engineered strain was finally obtained. The gene circuit design of this strain is theoretically capable of driving the expression of YopE1-15-fused PD-L1 nb protein under NIR light induction.

![Construction of the NIR Light-Induced YopE1-15-PD-L1 Nanobody Expression System Diagram](https://static.igem.wiki/teams/5922/results/img8.webp)

Figure 8: Construction of the PD-L1 Nanobody Expression System and Gene Circuit. (A) Plasmid map. The figure shows the plasmid map of the gene circuit used for PD-L1 nanobody (nb) expression. The core regulatory elements of the NETMAP system are all integrated into the pSB1A3 backbone. The reporter gene has been replaced with the sequence of YopE1-15-fused PD-L1 nb, whose expression is regulated by the PmrkA promoter. (B) Agarose gel electrophoresis of the YopE1-15-PD-L1 nb gene fragment. (C) Schematic diagram of the gene circuit of the NETMAP system.

### Conclusion

The PD-L1 nanobody expression system (DH5α-NETMAP-PDL1 nb) that can be precisely regulated by NIR light was successfully constructed. The construction of this system effectively combines the light-controlled remote sensing element (NETMAP) with the immunotherapeutic functional element (PD-L1 nb).

## Verification of NIR Light-Induced Expression and Extracellular Secretion of YopE1-15-PD-L1 Nanobody

### Objective

This experiment aimed to verify whether the light-controlled system based on the NETMAP promoter can successfully drive the expression of the PD-L1 nanobody (nb). Meanwhile, by detecting the presence of His-tag protein in the culture supernatant, it was confirmed that the target protein was successfully secreted outside the cells.

### Methods

Strain Activation and Cultivation: The DH5α-NETMAP-PDL1 nb engineered strain was inoculated at a ratio of 1:100 into 5 mL of LB medium containing 100 μg/mL Amp. A 15 mL centrifuge tube was wrapped with tin foil, and the strain was cultured at 37°C with shaking at 180 rpm for 12 hours. Subsequently, the bacterial cells were inoculated at a 1% rate into a 48-well plate containing 500 μL of Amp⁺ Cm⁺ LB medium and continuously cultured at 37°C with shaking at 180 rpm until the OD600 reached 0.5.

NIR Light Induction and Protein Sample Preparation: When the OD600 of the bacterial culture reached 0.5, a light-emitting diode (LED) lamp was used to initiate NIR light (660 nm) irradiation of the samples for 5 hours. Immediately after the induction, the supernatant of the engineered bacterial culture was collected. The supernatant was centrifuged at 12,000×g for 10 min at 4°C and then filtered through a 0.22 μm filter membrane to remove cells. Subsequently, the BeyoGold™ ultrafiltration tube (15 mL, 5 kDa MWCO, PES, FUF505, Beyotime) was used for protein concentration by centrifugation at 4,000×g for 40 min at 4°C. The protein concentration of the concentrated protein sample was determined using the Bradford kit (P0006, Beyotime).

Western Blot (WB) Analysis: The concentrated supernatant protein samples were separated by electrophoresis using a 12.5% SDS-PAGE gel prepared with the one-step PAGE gel rapid preparation kit (PG113, Yamei). Subsequently, the proteins were transferred to a 0.22 μm PVDF membrane (WJ001S, Yamei) via the wet transfer method. The membrane was blocked with protein-free rapid blocking solution (ED0024, Sikejie) at room temperature for 20 min. After blocking, the membrane was incubated with a 1:1000-diluted mouse His-tag antibody (AH367, Beyotime) (primary antibody) at 4°C overnight. Finally, the membrane was incubated with HRP-labeled goat anti-mouse IgG (H+L) (A0216, Beyotime) (secondary antibody) at room temperature for 1 hour. Chemiluminescence analysis was performed using a protein blotting imaging system (ChemiDoc MP, BioRad).

### Results

Western Blot (WB) analysis results showed that after 5 hours of induction with 660 nm NIR light, a clear specific band was successfully detected in the concentrated supernatant sample of the DH5α-NETMAP-PDL1 nb experimental group. The molecular weight of this band was consistent with the expected size of YopE1-15-PD-L1 nb.

![Verification of NIR Light-Induced Expression and Extracellular Secretion of YopE1-15-PD-L1 Nanobody Diagram](https://static.igem.wiki/teams/5922/results/img9.webp)

Figure 9: WB Experimental Results of YopE1-15-PD-L1 nb

### Conclusion

The NETMAP light-controlled system can successfully drive the expression of the PD-L1 nanobody target gene under the induction of 660 nm NIR light, and this protein can be effectively secreted outside the cells.

## System 2: Therapeutic System - Expression, Purification, and Activity Verification of Coagulase (Coa)

This study aimed to construct and verify the core effector molecule, Coagulase (Coa) protein, in the therapeutic system. First, efficient expression of the Coa gene was achieved in the E. coli BL21 strain via the pET28a induction system. Subsequently, the Coa protein was successfully purified using nickel column affinity chromatography, and its high purity and correct expression were verified by SDS-PAGE and Western Blot (WB). Then, the functional activity of the purified Coa protein was evaluated through in vitro coagulation assays. The results showed that the Coa protein exhibits significant coagulation activity, and the coagulation effect shows a clear concentration dependence (the 100% concentration group has the shortest coagulation time). This study successfully prepared and verified the Coa protein with biological activity, providing a key therapeutic payload for the subsequent induction of thrombosis by engineered bacteria at the tumor site.

## Expression, Purification, and WB Verification of Coagulase Protein

### Objective

This experiment aimed to achieve and purify the Coagulase (Coa) protein via the pET28a induction system and verify its expression and purity using SDS-PAGE and Western Blot, thereby providing a high-purity protein sample for subsequent in vitro coagulation function tests.

### Methods

For the preparation of recombinant strains, induced expression, and crude protein extraction: After codon optimization of the Coagulase-encoding gene and removal of restriction enzyme sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI), the gene was cloned into the pET28a (m) vector via the NdeI and XhoI restriction enzyme sites. The recombinant plasmid was transformed into competent E. coli BL21 strain by the heat shock method (42°C, 1 min). Transformants were screened on LB solid plates containing 100 μg/mL kanamycin (Kana) to obtain the recombinant engineered strain BL21-Coa. After overnight culture of the BL21-Coa strain in LB broth containing Kana, it was inoculated at a ratio of 1:100 into 10 mL of fresh medium and cultured at 37°C with shaking at 180 rpm until the OD600 reached 0.5-0.8. At this point, 0.5 mM IPTG inducer was added, and the culture temperature was adjusted to 16°C, with cultivation continued for 20 hours. After fermentation, the supernatant was discarded by centrifugation (5,000×g, 10 min), and the bacterial pellet was resuspended in 10 mL of PBS. Ultrasonic disruption was performed on ice using an ultrasonic disruptor (ultrasonic power: 70%; ultrasonic cycle: 1 s on, 3 s off; total ultrasonic time: approximately 15 min). Subsequently, centrifugation was conducted at 5,000×g for 10 min at 4°C, and the supernatant was collected as the crude protein sample. The concentration was determined using the Bradford kit. For Coa protein purification and WB verification: The protein purification kit (Beyotime, P2226) was used for nickel column affinity chromatography (Ni-NTA) purification to obtain purified Coa protein. The collected purified protein fractions were analyzed for purity by SDS-PAGE, and the expression of the target protein was verified by Western Blot: The protein samples were separated using a 12.5% SDS-PAGE gel prepared with the one-step PAGE gel rapid preparation kit. The proteins were transferred to a PVDF membrane, and after blocking with protein-free rapid blocking solution, the membrane was sequentially incubated with a 1:1000-diluted mouse His-tag antibody (primary antibody) and HRP-labeled goat anti-mouse IgG secondary antibody. Finally, chemiluminescence analysis was performed using a protein blotting imaging system. The concentration of the final purified protein was determined using the Bradford kit.

![Expression, Purification, and WB Verification of Coagulase Protein Diagram](https://static.igem.wiki/teams/5922/results/img10.webp)

Figure 10: Construction of the Coagulase Expression System and Gene Circuit. (A) Plasmid map. The figure shows the plasmid map of the gene circuit used for Coa expression. (B) Agarose gel electrophoresis of the Coa gene fragment. (C) Schematic diagram of the Coa system gene circuit.

### Results

SDS-PAGE and Western Blot results showed that after IPTG induction, low-temperature cultivation, and nickel column affinity chromatography, the purified Coa protein exhibited a clear and single main band on the gel, consistent with the expected molecular weight. WB results confirmed the successful expression and purification of the target protein.

![Expression, Purification, and WB Verification of Coagulase Protein Diagram](https://static.igem.wiki/teams/5922/results/img11.webp)

Figure 11: WB Analysis of Bacterial Lysates from E. coli BL21 Carrying the pET28a Recombinant Vector After Induction with 0.5 mM IPTG at 16°C for 20 Hours

### Conclusion

The Coagulase protein was successfully expressed via the pET28a system and purified to high purity through nickel column affinity chromatography, providing a qualified sample for subsequent in vitro coagulation function verification.

## Verification of Coagulase Coagulation Activity

### Objective

This experiment aimed to evaluate the coagulation activity of purified Coagulase (Coa) protein at different concentrations when mixed with blood samples through in vitro coagulation assays, so as to determine the functional effectiveness of the Coa protein and its concentration dependence.

### Methods

Non-anticoagulated blood samples were used. Subsequently, 50 μL of purified Coa protein solutions with different concentrations were mixed with 50 μL of blood samples in EP tubes or 96-well plates, with a total reaction volume of 100 μL. The concentration gradient of the Coa protein solution included 100%, 80%, 60%, 40%, and 20%, and a mixture of 0% Coa solution (i.e., buffer) and 50 μL of blood sample was used as the negative control. Meanwhile, 100 μL of water and 100 μL of 100% Coa solution were set as blank controls without blood. All mixtures were incubated in a 37°C incubator, and photos were taken every 5 minutes to record the coagulation status for 30 minutes. A final photo was taken at 60 minutes when complete coagulation was achieved. The main observation and analysis indicators included thrombosis formation time, thrombus morphology, and coagulation intensity.

### Results

The Coa protein exhibited concentration-dependent coagulation activity: the 100% and 80% concentration groups showed the fastest coagulation rate, with stable thrombus formation expected within 5-10 minutes and the highest coagulation intensity. As the Coa concentration decreased gradiently, the coagulation time was significantly prolonged. The coagulation rate and intensity of the negative control group (0% Coa) were much lower than those of the Coa experimental groups.

![Verification of Coagulase Coagulation Activity Diagram](https://static.igem.wiki/teams/5922/results/img12.webp)

Figure 12: Coagulation Experiment

### Conclusion

The in vitro coagulation assay results confirmed that the purified Coagulase protein has coagulation activity, and its coagulation effect is positively correlated with the protein concentration. This supports the use of Coa protein as an effective payload for inducing thrombosis in the targeted therapy of engineered bacteria.

# System 3: Construction and Functional Verification of the Targeting System

To achieve specific targeting of engineered probiotics to colorectal cancer cells, this study constructed an engineered strain with surface-displayed HlpA. Using a truncated ice nucleation protein (INP) as the anchoring motif, the HlpA fusion protein (INP-HlpA-mRFP) was stably displayed on the surface of E. coli. Its expression was driven by a constitutive promoter (J23100-B0034), and mRFP fluorescent protein was coupled for visualization. The pET28a induction and Western Blot experiments successfully verified the molecular weight and expression of the INP-HlpA target protein. Functional verification experiments showed that this engineered strain (BL21-INP-HlpA-mRFP) exhibits a significant adhesion effect on CT26 colorectal cancer cells. The adhesion efficiency was confirmed by CFU counting and fluorescence imaging, verifying that this targeting system can achieve targeted enrichment at the site of target cancer cells.

## Expression and Western Blot Verification of the INP-HlpA Targeting Protein

### Objective

This experiment aimed to achieve efficient expression of the INP-HlpA-encoding gene in the E. coli BL21 strain via the pET28a induction system and verify the correct molecular weight of the target protein by Western Blot (WB).

### Methods

Construction of Recombinant Plasmids and Preparation of Engineered Strains: The INP−HlpA-encoding gene was synthesized and codon-optimized for E. coli. Restriction enzyme sites (EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI) were removed to meet the RFC#10 standards and pET28a (m) cloning requirements. The INP-HlpA-encoding gene was cloned into the pET28a (m) vector via the NdeI and XhoI restriction enzyme sites. The recombinant plasmid was transformed into E. coli BL21 by the heat shock method, and positive clones were screened on LB solid plates containing 100 μg/mL kanamycin (Kana) to obtain the recombinant engineered strain BL21−INP−HlpA.

Induced Expression and Collection of Proteins: The BL21-INP-HlpA strain was cultured overnight in LB broth containing Kana. Subsequently, it was inoculated at a 1% ratio into 30 mL of fresh LB broth and cultured until the OD600 reached 0.2, at which point 0.5 mM IPTG was added to induce expression. The culture temperature was adjusted to 16°C, and cultivation was continued for 20 hours.

Extraction of Intracellular Proteins and WB Verification: 5 mL of the fermentation broth was centrifuged to collect the bacterial cells, which were then resuspended in PBS. Ultrasonic disruption was performed on ice using an ultrasonic disruptor. After centrifugation of the lysate, the supernatant was collected as the intracellular protein sample, and its concentration was determined using the Bradford kit.

Western Blot (WB) Analysis: The intracellular protein samples were separated using a 12.5% SDS-PAGE gel, and transferred to a PVDF membrane via the wet transfer method. After blocking the membrane, it was sequentially incubated with a 1:1000-diluted mouse His−tag antibody (primary antibody) and HRP-labeled goat anti-mouse IgG (secondary antibody). Finally, chemiluminescence analysis was performed using a protein blotting imaging system.

![Expression and Western Blot Verification of the INP-HlpA Targeting Protein Diagram](https://static.igem.wiki/teams/5922/results/img13.webp)

Figure 13: Construction of BL21-INP-HlpA. (A) The plasmid map of pET-28a(m)-INP-HlpA. (B) The gene circuit of BL21-INP-HlpA.

### Results

Western Blot analysis results showed that after IPTG induction and low-temperature cultivation for 20 hours, a clear specific band was successfully detected in the intracellular protein sample of the BL21-INP-HlpA strain. The molecular weight of this band was consistent with the expected molecular weight of INP-HlpA and was specifically recognized by the His-tag antibody. (See the figure: WB Analysis of Bacterial Lysates from E. coli BL21 Carrying the pET28a Recombinant Vector After Induction with 0.5 mM IPTG at 16°C for 20 Hours)

![Expression and Western Blot Verification of the INP-HlpA Targeting Protein Diagram](https://static.igem.wiki/teams/5922/results/img14.webp)

Figure 14: WB Analysis of Bacterial Lysates from E. coli BL21 Carrying the pET28a Recombinant Vector After Induction with 0.5 mM IPTG at 16°C for 20 Hours

### Conclusion

The INP-HlpA-encoding gene achieved efficient induced expression in the BL21 strain via the pET28a system. Western Blot verified the successful expression and correct molecular weight of the target protein.

## Construction of the Targeting Strain

### Objective

This experiment aimed to construct an engineered strain carrying the INP-HlpA surface display system to constitutively display the colorectal cancer cell-specific recognition protein HlpA on the surface of E. coli and realize the visual tracking of this strain using the mRFP tag.

### Methods

INP was fused to the N-terminus of HlpA to serve as the anchoring motif for cell surface display, and mRFP was coupled downstream of HlpA to enable visual observation of the adhesion effect. The INP-HlpA-mRFP-encoding gene was synthesized (Generalbiol, China) and codon-optimized for E. coli. Restriction enzyme sites (EcoRI, XbaI, SpeI, and PstI) were removed to meet the RFC#10 standards. The constitutive promoter J23100-B0034 (J23100-B0034 cis-acting element) was introduced upstream of INP-HlpA to drive stable expression. Subsequently, this expression cassette was cloned into the pSB1A3 vector via the XbaI and SpeI restriction enzyme sites. The recombinant plasmid was transformed into competent E. coli BL21 strain by the heat shock method (42°C, 1 min). Transformants were screened on LB solid plates (supplemented with 1.5% agar) containing 100 μg/mL ampicillin (Amp) and verified by sequencing (Tsingke, Beijing), finally obtaining the recombinant engineered strain BL21-INP-HlpA-mRFP. The engineered strain was stored in a culture medium containing 25% (v/v) glycerol (-20°C). Its regular culture conditions were 37°C with shaking at 150 rpm, and inoculation and expansion culture were performed using LB broth containing 100 μg/mL Amp.

![Construction of the Targeting Strain Diagram](https://static.igem.wiki/teams/5922/results/img15.webp)

Figure 15: Construction of the INP-HlpA Expression System and Gene Circuit. (A) Plasmid map. The figure shows the plasmid map of the gene circuit used for INP-HlpA expression. (B) Agarose gel electrophoresis of the INP-HlpA gene fragment. (C) Schematic diagram of the INP-HlpA system gene circuit.

### Results

The results confirmed that the INP-HlpA-mRFP expression cassette was correctly inserted into the pSB1A3 vector, and the promoter J23100-B0034 could drive the stable expression of this gene. The engineered strain BL21-INP-HlpA-mRFP exhibited stable mRFP red fluorescence under regular culture conditions, initially confirming the successful expression of the adhesion protein INP-HlpA and the construction of the surface display system.

### Conclusion

The BL21-INP-HlpA-mRFP engineered strain was successfully constructed. This strain can constitutively display the HlpA adhesion protein on the cell surface and emit mRFP red fluorescence.

## Adhesion Effect Test of the INP-HlpA Engineered Strain on Colorectal Cancer CT26 Cells

### Objective

This experiment aimed to evaluate the specific adhesion efficiency of the engineered strain with surface-displayed HlpA (BL21-INP-HlpA-mRFP) to colorectal cancer CT26 cells through in vitro co-culture and quantitative CFU counting.

### Methods

2×10⁵ CT26 cells were inoculated into a 6-well cell culture plate and cultured for 48 hours until the cell confluence reached 80%. Subsequently, the medium was replaced with fresh DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 50 mg/L Amp. Next, 1×10⁷ CFU of the BL21-INP-HlpA-mRFP engineered probiotic was inoculated and co-cultured with CT26 cells for 2 hours. After co-culture, 100 μL of the supernatant was collected, diluted, and spread on Amp-containing plates. The number of free bacteria was quantified by the CFU counting method, and the number of bacteria adhered to the cells and the adhesion efficiency were calculated accordingly. Subsequently, the CT26 cells were washed twice with sterile PBS to remove non-adherent bacteria. Finally, an inverted fluorescence microscope was used to capture mRFP fluorescence images to visually observe the distribution and adhesion morphology of the bacteria on the surface of CT26 cells.

### Results

CFU counting results showed that the BL21-INP-HlpA-mRFP strain had a high adhesion efficiency, confirming the specific adhesion effect mediated by HlpA. Images captured by the inverted fluorescence microscope clearly showed that a large number of engineered bacteria labeled with mRFP adhered tightly to the surface of CT26 cells. The results of adhesion rate calculation via CFU counting showed that the adhesion efficiency of the BL21-INP-HlpA-mRFP strain was significantly higher than that of the control group. This result confirms the specific adhesion effect mediated by HlpA, and this engineered strain can efficiently target and adhere to CT26 colorectal cancer cells.

![Adhesion Effect Test of the INP-HlpA Engineered Strain on Colorectal Cancer CT26 Cells Diagram](https://static.igem.wiki/teams/5922/results/img16.webp)

Figure 16: Adhesion Effect Test. Figure (1) Adhesion effect diagram of the BL21-INP-HlpA-mRFP engineered strain on CT26 colorectal cancer cells. Figure (2) Comparison of adhesion rates of the engineered strain to CT26 cells.

(scale bar: 5 μm).

### Conclusion

The BL21-INP-HlpA-mRFP engineered strain successfully achieved specific adhesion to CT26 colorectal cancer cells. The quantitative results of adhesion efficiency and direct evidence from mRFP fluorescence images collectively confirm the functionality of the INP-HlpA surface display system, laying a foundation for the engineered strain to achieve targeted localization at the tumor site.

# System 4: Construction and Evaluation of the Biosafety Suicide System

This study aimed to construct a highly controllable biosafety suicide system, with the core being the use of the potent bacterial toxin protein MazF to achieve self-clearance of engineered bacteria under specific conditions. The initial design adopted the lactose-inducible promoter Plac and its repressor protein LacI to regulate the expression of MazF. However, no positive clones could be obtained during the transformation and screening process of this construct. The main reason is that the Plac promoter has unavoidable low-level leaky expression, and the MazF toxin produced by leakage is sufficient to kill all successfully transformed host cells. This proves that the Plac/LacI system lacks the necessary tightness for the control of highly toxic genes. To address this fatal flaw, the study turned to the arabinose-inducible promoter PBAD with a stronger inhibitory effect, successfully constructing the PBAD−MazF plasmid and obtaining a stably heritable recombinant engineered strain DH5α−PBAD−MazF. This successful construction confirms the effective inhibition of the PBAD system on the leaky expression of MazF under non-inducing conditions. Subsequent functional tests verified the system performance by monitoring the growth curve: under arabinose-free (non-inducing) conditions, the strain showed normal growth, verifying the high safety of the system; under arabinose-added (inducing) conditions, the expression of MazF toxin is expected to cause rapid growth arrest or severe growth inhibition of the strain. In conclusion, the MazF suicide system based on PBAD is a feasible and efficient biosafety solution, which can achieve precise and tight control over the fate of engineered bacteria through external inducers.

## Construction of the Suicide System Based on the Plac Promoter

### Objective

To initially construct a MazF toxin safety system based on the lactose-inducible promoter Plac and the LacI repressor protein, and evaluate its ability to control cytotoxicity under non-inducing conditions.

### Methods

The MazF toxin gene was synthesized and codon-optimized. The MazF gene was placed downstream of the Plac promoter, and a LacI expression element (PlacI−LacI) was inserted upstream to construct the PlacI−LacI−B0035−Plac−mazF sequence. This sequence was cloned into the pSB1A3 vector and transformed into E. coli DH5α, and an attempt was made to screen positive clones in a medium containing Amp.

![Construction of the Suicide System Based on the Plac Promoter Diagram](https://static.igem.wiki/teams/5922/results/img17.webp)

Figure 17: Construction of the Plac−mazF Suicide System. A: Plasmid construction map of the Plac−mazF suicide system. B: Gene circuit diagram of the Plac−mazF suicide system construction.

### Results

No positive clones could be screened out.

### Conclusion

The Plac promoter has unacceptable low-level leaky expression. Due to the strong toxicity of MazF, even with the inhibitory attempt of the LacI protein, the toxin produced by leakage is sufficient to kill all successfully transformed host cells carrying the plasmid, indicating that the Plac/LacI system is not suitable for the safe control of highly toxic genes.

## Construction of the Suicide System Based on the PBAD Promoter

### Objective

To address the defects of the Plac system, the arabinose-inducible promoter (PBAD) was introduced as a replacement to construct a new MazF safety system, and its tight regulatory ability under non-inducing conditions (i.e., allowing strain survival) was verified.

### Methods

The codon-optimized MazF toxin gene was used. The MazF gene was placed downstream of the PBAD promoter to construct the PBAD−mazF sequence. The PBAD−mazF was cloned into the pSB1A3 vector. The recombinant plasmid was transformed into E. coli DH5α by the heat shock method. Positive clones were screened on solid plates containing 100 μg/mL ampicillin (Amp) and verified by sequencing.

![Construction of the Suicide System Based on the PBAD Promoter Diagram](https://static.igem.wiki/teams/5922/results/img18.webp)

Figure 18: Construction of the PBAD−MazF Suicide System. A: Plasmid construction map of the PBAD−MazF suicide system. B: Gene circuit diagram of the PBAD−MazF suicide system construction.

### Results

Positive clones were successfully screened, and the recombinant engineered strain DH5α−PBAD−MazF was obtained.

### Conclusion

The suicide system based on the PBAD promoter was successfully constructed. This confirms that the PBAD promoter has a much better inhibitory effect on the leaky expression of the MazF toxin gene under non-inducing conditions (without arabinose) compared to Plac, meeting the tight regulatory standards required for the construction and expansion of engineered strains.

## Functional Test of the PBAD−MazF Suicide System

### Objective

To quantitatively evaluate the function of the PBAD−MazF system under conditions with and without the inducer (arabinose) by monitoring the growth curve, and verify its leak-free safety and induced lethality efficiency.

### Methods

The frozen DH5α−PBAD−MazF strain was activated and inoculated at a ratio of 1:100 into LB medium containing Amp, and the OD600 was adjusted to 0.1. The culture was divided into two groups: the induction group, to which 2% arabinose was added (to induce MazF expression); and the control group, without arabinose addition. At 37°C, a FlexStation 3 was used to continuously monitor the OD600 of the strains in both groups and record the growth curves.

### Results

The control group strain showed normal growth. The growth curve of the induction group strain showed severe growth inhibition or complete growth arrest.

![Functional Test of the PBAD−MazF Suicide System Diagram](https://static.igem.wiki/teams/5922/results/img19.webp)

Figure 19: Growth Curves of the Control Group and Induction Group Strains

### Conclusion

The normal growth of the control group confirms the high safety (extremely low leakage) of the PBAD system in the non-inducing state. The growth arrest or inhibition of the induction group confirms the efficient lethality of the MazF toxin, thereby comprehensively verifying that the PBAD−MazF construct is a fully functional and controllable biosafety suicide system.

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