iGEM 2025 — Team smu-union-china

- [*![](https://static.igem.wiki/teams/5840/figure/background.webp)
  OVERALL SCHEME*](#Overall Scheme)

- [*![](https://static.igem.wiki/teams/5840/figure/future-work.webp)
  WEEKLY TASK
              ARRANGEMENT*](#Weekly Task Arrangement)

- [*![](https://static.igem.wiki/teams/5840/figure/ret-4.webp)
  WET LAB DETAILED
                  TIMETABLE*](#Wet Lab Detailed Timetable)

- [*![](https://static.igem.wiki/teams/5840/figure/inspiration.webp)
  IMAGE GALLERY OF
              THE EXPERIMENTS*](#Image Gallery of the Experiments)

## Overall Scheme

Our notebook documents the entire journey of our team throughout the 2025 iGEM season. It records our daily activities, experiments, discussions, and decisions in chronological order. This detailed record reflects both our scientific process and our teamwork.

|  | Week1 | Week2 | Week3 | Week4 | Organizer/ Participator |
| --- | --- | --- | --- | --- | --- |
| January | Winter Holiday - Post recruitment information | | | | Ke Liu |
| February | Team Formation - In-person interview | | | | Ke Liu, Ling Li,Chenguang Shen |
| March | • Group cooperative learning  • Research on the iGEM Website | • Select the track and theme; Brainstorming;Designed expertments【Wetlab & Drylab】  • Integrate existing resources; Built the HP framework【HP】  • Select the design style; Design the team flag and emblem【Art Group】  • Learn how to build a web framework by referring to the best wiki teams【Wiki】 | | • Brainstorming; Determine the specific parts and Protocols【Wetlab & Drylab】  • Refine the work details in the directions of ihp, education, and inclusivity【HP】  • Modify the PPT template【Art Group】  • Learn front-end technology【Wiki】 | All team members |
| April | • Obtain chassis bacterial; Purchase experimental consumables【Wetlab】  • Epidemiological investigation model【Drylab】  • Design interview outlines for clinicians, patients (and their families); Design questionnaires【HP】  • Picture book and short video production; Design iP and wiki animation【Art Group】  • Learn Mini Program game development【Wiki】 | | • Guangzhou Regional Exchange Conference of iGEM Greater Bay Area Synthetic Biology Industry-Academia-Research Forum Successfully Held at Southern Medical University  • Plasmid construction;Prepare for on-site report;Consulting expert【Wetlab】  • Model fitting【Drylab】  • Interviews Clinical doctor/Patient (and family member)/Enterprise; Promotion of Rural Science Popularization Work in Zhaoqing【HP】  • wiki Page design;Prepare the roller banner and peripherals【Art Group】  • Mini Program game development【Wiki】 | | All team members |
| May | • The initially selected protein structure of H1N1; Protein-protein docking prediction【Wetlab & Drylab】  • Synthetic Biology Open Day Event; Science experiment kit; Popular Science Video Proposal; Colorblind and blind-friendly design; Planning in the direction of women's rights protection【HP】  • Design wiki page【Art Group】  • AR picture book;Complete the web page【Wiki】 | • Preliminary Project Safety Form; Village Selection; Growth curve plotting【Wetlab】  • AlphaFold constructs the structure of the medi8852 antibody; extracts docking energy【Drylab】  • Second Round Biosynthesis Open Day; Science popularization video making; Synchronously update activity records on various media platforms【HP】  • Excellent video analysis for Project Promotion Video; wiki page design【Art Group】  • Wiki page development【Wiki】 | • The 9th iGEM South China Exchange Conference  • Primer Ordering; Project description; Consumables Confirmation & Order Placement【Wetlab】  • medi8852 scfv sequence ligand construction of 3D model; Protein docking of medi8852 and h1n1【Drylab】  • Interviews; Advance Women's Rights Protection (Questionnaire Collection and Interviews)【HP】  • Design team uniforms; Collection and production of creatives for the project promotion video【Art Group】 | | All team members |
| June | • Electroporation transformation【Wetlab】  • Try rigid docking, zdock; Find the literature docking checkpoint and further perform flexible docking【Drylab】  • Improve the HP framework; Questionnaire Collection and Data Analysis; Phase III Open Day Collaboration; Proposals to improve the needs of international students【HP】  • Production of iGBA Conference Pass (Seed Paper); Promotion video script【Art Group】  • Upload wiki; fix bugs【Wiki】 | | • Contribution Planning; CCIC presentation PPT; shoot a promotion video【Wetlab】  • Optimize zdock modeling - mdockpp Calculate docking dynamics; Fitted growth curve OD value【Drylab】  • Science popularization tweet; Hundred-Thousand-Million Project; Interview with an obstetrician-gynecologist【HP】  • Poster production; Design a pregnancy epidemic prevention kit; Pregnancy Handbook Production 【Art Group】  • Upload wiki; fix bugs【Wiki】 | | All team members |
| July | • Summer iGBA Forum: Where Innovation Meets Responsibility  • Write the parts content; Design the safety content; Electroporation & PCR【Wetlab】  • IHP feedback mechanism; Organize the text and pictures of the activities that have been carried out【HP】  • Promotion video editing; animation production; AR picture book layout; homepage production; Create visual charts and their animations【Art Group】  • Upload wiki; fix bugs【Wiki】 | | • Safety & Security Page; Part documentation completed; Design Engineering【Wetlab】  • seiar model【Drylab】  • Organize the list of stakeholders; attribution; team members information collection; Questionnaires for medical students, patients; education/inclusivity wiki page; Organize the results of legislative direction【HP】  • Edit promotion video; Create wiki creatives【Art Group】  • Upload wiki; fix bugs【Wiki】 | | All team members |
| August | • The 12th China Conference on iGEM (CCiC)  • Parts Upload; Improve the Contribution page; Write the design interface【Wetlab】  • Calculate the transmission rate of antibody use based on antibody IC50; Calculate the changes in the solubility curve of scFv at different pH values; Rationality of applying CCD modeling to predict different pH/temperature reaction interfaces in LGG engineered bacteria【Drylab】  • Organize creatives and design the ihp-wiki page; Popular science series work; Community street stall promotion; Women-themed interview 【HP】  • Three-school picture book collaboration; wiki design【Art Group】  • Upload wiki; fix bugs【Wiki】 | | • Check-in form; Final safety form; Electroporation & PCR【Wetlab】  • Randomization added to SEIAR model parameter modification【Drylab】  • Street vending in Tianhe District; Organize collaboration; National University of Defense Technology White Paper; Women's rights program advancement 【HP】  • Presentation video pre-production/brainstorming; Team members page design; Popular Science Tweet Series, Issue 6【Art Group】  • Layout of the home page; Code of the member page; Sidebar; Text; Input for other pages【Wiki】 | | All team members |
| September | • Changes in bacteria under different virus titers with viral load as a variable; PCR; electroporation of two plasmids; Virus incubation for fluorescence measurement; Temperature-induced detection of EGFP expression; Expression of light-GFP【Wetlab】  • RNA thermometer design; write wiki page,Project Software【Drylab】  • Guangzhou Library Lecture; write wiki page;Multilingual (translated picture book)【HP】  • Design wiki page【Art Group】  • Upload wiki; fix bugs【Wiki】 | | • Supplementary Experiments  • Project Software  • Wiki Design and Documentation  • Wiki Upload  • Judging Form | | All team members |
| October | • Wiki Design and Documentation  • Wiki Upload  • Judging Form  • Presentation Video | • Presentation Video upload | • Defense Preparation | • Participation in the 2025 Grand Jamboree |  |

## Weekly Task Arrangement

Between March and October, our team leader, Ke Liu, coordinated weekly meetings with each subgroup. During these sessions, the specific tasks for the following week were discussed and documented. The shared work document ensured transparency and collaboration, as all team members could access, edit, and upload their progress. This systematic approach to project management helped the team maintain clear communication, effective coordination, and steady progress throughout the season

#### Weekly task records (PDF uploads):

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## Wet Lab Detailed Timetable

In addition to the overall scheme, a more detailed timetable for the wet lab stage of the project can be found below.

![](https://static.igem.wiki/teams/5840/figure/notebook-p1.webp)

## Image Gallery of the Experiments

![](https://static.igem.wiki/teams/5840/figure/notebook-p2.webp)
![](https://static.igem.wiki/teams/5840/figure/notebook-p3.webp)

![](https://static.igem.wiki/teams/5840/figure/notebook-p4.webp)

![](https://static.igem.wiki/teams/5840/figure/notebook-p7-1-1-1.webp)

![](https://static.igem.wiki/teams/5840/figure/notebook-p8-1.webp)

![](https://static.igem.wiki/teams/5840/figure/notebook-p5.webp)

- [*![](https://static.igem.wiki/teams/5840/figure/ent-1.webp)
  SENSING MODULE*](#Sensing Module)

- [*![](https://static.igem.wiki/teams/5840/figure/ent-2.webp)
  RESPONSE MODULE*](#Response Module)

- [*![](https://static.igem.wiki/teams/5840/figure/ent-3.webp)
  SAFETY MODULE*](#Safety Module)

- [*![](https://static.igem.wiki/teams/5840/figure/ent-4.webp)
  FINAL CYCLE*](#final cycle)

![](https://static.igem.wiki/teams/5840/figure/cyc1-1.webp)

![](https://static.igem.wiki/teams/5840/figure/cyc2-1.webp)

![](https://static.igem.wiki/teams/5840/figure/cyc2-1.webp)

![](https://static.igem.wiki/teams/5840/figure/cyc2-1.webp)

![](https://static.igem.wiki/teams/5840/figure/cyc1-1.webp)

![](https://static.igem.wiki/teams/5840/figure/cyc2-1.webp)

![](https://static.igem.wiki/teams/5840/figure/cyc2-1.webp)

![](https://static.igem.wiki/teams/5840/figure/cyc2-1.webp)

## Safety Module

### Initial Concerns

* 1. There was a risk that engineered bacteria could survive and replicate outside the body, potentially causing environmental harm if they were excreted and entered natural ecosystems.
* 2. Should the engineered bacteria migrate from the nasal cavity to the lower respiratory tract, they could potentially trigger infections or other detrimental effects, attributable to their engineered characteristics.
* 3. When engineered probiotics, intended for human application, do trigger undesirable clinical risks—albeit with a minuscule probability, what measures can be promptly implemented to curb these risks?
* 4. The practicality of our design must also consider the stability and safety of the engineered bacteria during transportation and storage, as well as their compatibility with potential delivery methods.

![](https://static.igem.wiki/teams/5840/figure/cycle17.webp)

![](https://static.igem.wiki/teams/5840/figure/cycle17.webp)

![](https://static.igem.wiki/teams/5840/figure/cycle17.webp)

## Improvement selection strategy

### stabilize plasmids while reducing growth burden

![](https://static.igem.wiki/teams/5840/figure/cycle17.webp)

- [*![](https://static.igem.wiki/teams/5840/figure/lgg-growth-curve.webp)
  LGG GROWTH
             CURVE
             DETERMINATION*](#LGG)

- [*![](https://static.igem.wiki/teams/5840/figure/electroporation.webp)
  ELECTROPORATION
             TRANSFORMATION*](#Electroporation)

- [*![](https://static.igem.wiki/teams/5840/figure/stored-procedure.webp)
  COLONY PCR*](#Colony)

- [*![](https://static.igem.wiki/teams/5840/figure/fluorescent-verification.webp)
  FLUORESCENT
             VERIFICATION*](#Fluorescent)

- [*![](https://static.igem.wiki/teams/5840/figure/influenza-virus-ha.webp)
  INFLUENZA VIRUS HA
             HEMAGGLUTINATION
             ASSAY*](#Influenza)

- [*![](https://static.igem.wiki/teams/5840/figure/micro-neutralization.webp)
  ELISA*](#Micro-neutralization)

- [*![](https://static.igem.wiki/teams/5840/figure/cell-lysis-procedure.webp)
  BACTERIAL CELL
             MEMBRANE LYSIS*](#Cell)

- [*![](https://static.igem.wiki/teams/5840/figure/heat-induced-rna.webp)
  GFP EXPRESSION*](#Heat-Induced)

- [*![](https://static.igem.wiki/teams/5840/figure/heat-induced-rna.webp)
  GFP EXPRESSION*](#Heat-Induced RNA (ROSE)-Controlled GFP Expression)

- [*![](https://static.igem.wiki/teams/5840/figure/small-molecule.webp)
  PROMOTER EXPRESSION*](#Small)

- [*![](https://static.igem.wiki/teams/5840/figure/functional-verification.webp)
  BLUE LIGHT AND
             NATURAL LIGHT
             INDUCTION*](#Functional Verification of Blue Light & Natural Light Induction)

- [*![](https://static.igem.wiki/teams/5840/figure/function-verification-of-the-repressor-protein.webp)
  REPRESSOR PROTEIN
             INDUCTION*](#Functional)

## LGG Growth Curve Determination

### Ⅰ. Basic Principles

When bacteria are inoculated into a liquid medium and the cell number is counted at regular intervals, a typical bacterial growth curve can be plotted, showing four distinct growth phases of bacteria over time.

- Lag Phase: Slow or lack of growth due to the physiological adaptation of cells to culture conditions, or slow growth due to low initial cell density.
- Log or Exponential Phase: The optimal growth rate is achieved, during which the cell number increases logarithmically over discrete time intervals.
- Stationary Phase: Bacterial growth and death balance each other, resulting in no net increase in cell number. The decline in growth rate is typically due to nutrient depletion or accumulation of toxic waste components.
- Decline or Death Phase: The death rate exceeds the growth rate, leading to a net loss of viable cells.

The turbidimetric method can be used to plot the growth curve of bacteria in broth or liquid medium. It is one of the simplest methods for analyzing growth trends. As the cell number in the sample increases, the transmission of light through the sample decreases. A spectrophotometer is used to track the changes in optical density over time.

### Ⅱ. Experimental Materials and Preparation

1. Strain: Lactobacillus rhamnosus.

2. Culture media: MRS broth culture medium, MRS agar culture medium.

3. Instruments: sterile culture flasks, test tubes, constant temperature shakers or constant temperature incubators, spectrophotometers, pipettes, sterile pipettes, centrifuge tubes, etc.

### Ⅲ. Experimental Procedures

1. Inoculation and Cultivation:

- [Day 1] Revive bacteria, use a sterile loop to inoculate the bacteria onto an agar plate, and incubate at 37°C for 18-24 hours.
- [Day 2] Pick a single colony from the MRS agar plate and inoculate it into a test tube containing 10 ml of autoclaved broth medium. Incubate the test tube at 37°C overnight.
- [Day 3] Inoculate 0.1% overnight culture into a test tube containing 10 ml of autoclaved broth medium. Measure the OD value at 0 hours. Incubate at 37°C. Take a certain amount of the culture suspension every 1 hour and measure the optical density at a wavelength of 600 nm using a spectrophotometer until the reading becomes static.

2. Data Recording:

At the end of the experiment, plot the time in minutes on the X-axis against the optical density at 600 nm on the Y-axis to obtain the growth curve of bacteria. Repeat the experiment at least 3 times to reduce the error.

### Ⅳ. Data Processing and Curve Plotting

1. Data Organization: Calculate the mean and standard deviation for each time point. If using OD values, confirm that they exhibit a linear relationship with bacterial concentration.

2. Curve Plotting: Use time as the abscissa (X-axis) and the logarithm of the OD value or the viable cell count as the ordinate (Y-axis). Plot the growth curve using software such as Excel, GraphPad Prism, or Origin.

3. Curve Analysis:

- Lag Phase: Bacteria adapt to the environment; the OD value changes slowly.

- Log Phase: Bacteria proliferate rapidly; the OD value increases exponentially.

- Stationary Phase: The bacterial population reaches its maximum; the OD value stabilizes.

- Death Phase: Bacteria die; the OD value declines.

### Ⅴ. Precautions

1. Aseptic Technique: Ensure sterile procedures during inoculation and cultivation to avoid contamination.

2. Sampling Consistency: Maintain consistent sample volumes and time intervals during sampling.

3. Instrument Calibration: Calibrate the spectrophotometer in advance to ensure accurate OD measurements.

4. Experimental Repeats: Repeat the experiment at least three times to improve data reliability.

### Ⅵ. Frequently Asked Questions and Solutions

1. Large Fluctuations in OD Values: Check for uniform sampling and proper instrument calibration. Increase the number of replicates to reduce error.

2. Abnormal Growth Curve: Check if culture medium composition, temperature, pH, and other conditions are suitable. Confirm whether the bacterial strain is contaminated or has mutated.

3. Premature Onset of Death Phase: Check if nutrients are depleted and whether supplementation is needed. Verify that the culture conditions are suitable for bacterial growth.

## Electroporation Transformation

### Ⅰ. LGG Competent Cell Preparation

**Method 1:**
1. Inoculate the activated bacterial strain into MRS medium and culture at 37°C, 200 rpm for 8 hours.
2. Subculture at a 1% inoculation ratio into SMRS medium containing 2% glycine. Culture at 37°C, 200 rpm for 8 hours.
3. Centrifuge at room temperature, 10000 rpm for 3 minutes to collect the bacterial cells.
4. Wash the cell pellet with SMM buffer.
5. Resuspend and enzymatically digest the cells using SMM buffer containing 30 mg/mL lysozyme at 37°C for approximately 30 minutes.
6. After digestion, wash the cells thoroughly 1-2 times with pre-chilled 15% glycerol.
7. Resuspend the cells in pre-chilled 15% glycerol, aliquot 80 μL per tube, and store at 4°C or -80°C for later use.

**Method 2:**
1. Resuspend 12 ml of an activated culture in the logarithmic growth phase in 1 ml of ice-cold ultrapure water, and wash twice.
2. Resuspend the cells in 1 ml of 50 mM EDTA and keep on ice for 5 minutes.
3. Wash once in ultrapure water.
4. Wash twice in 0.3 M sucrose solution.
5. Resuspend in 360 μl of 0.3 M sucrose and temporarily store at 4°C for subsequent electroporation.

### Ⅱ. Plasmid Transformation via Electroporation

Note: Maintain low-temperature conditions on ice during electroporation
1. Take 80 μL of competent cells, add 1-2 μL of plasmid DNA (concentration 1 μg/μL), mix gently, and incubate on ice for 5 minutes.
2. Transfer the mixture into a pre-chilled 1 mm electroporation cuvette. Perform electroporation under the following conditions: Electric field strength 2 KV/cm, Resistance 200 Ω, Capacitance 25 μF.
3. Immediately after the pulse, add 1 mL of pre-chilled MRS broth (without Tween 80), mix gently, and incubate on ice for 5 minutes. Then, incubate at 28°C for 3 hours.
4. Spread 200/100 μL of the electroporated culture onto MRS solid medium plates containing erythromycin. Incubate at 37°C. Once single colonies appear, pick them for PCR verification.
5. As a negative control for colony PCR, spread competent cells that did not undergo electroporation onto a plate and pick single colonies after they grow.

### Ⅲ. Precautions

Electroporation Cuvette Reuse and Cleaning

1. Post-Use Cleaning of Electroporation Cuvettes:

a. Water Rinse: After use, rinse the cuvette extensively with tap water to remove residual bacterial culture and other matter. Then, rinse multiple times with deionized water to replace the tap water.
Alternative: Perform ultrasonic cleaning with ultrapure water for about 30 minutes, twice, to remove contaminants from the cuvette gap.
b. Ethanol Soak: Soak the cuvette in a 75% ethanol solution for about 2 hours for initial sterilization.
Alternative: Perform ultrasonic cleaning with 75% ethanol for 30 minutes, twice.
c. Water Rinse: Remove the cuvette and rinse it with deionized water at least 3 times. Then, use a 1 mL pipette to repeatedly aspirate and dispense ddH₂O into the cuvette chamber more than 10 times. Shake dry.
d. Dehydration with Absolute Ethanol: Fill the cuvette with absolute ethanol for dehydration.
e. Storage: Air-dry thoroughly under a laminar flow hood, place in a sterile container, and store at -20°C (to prevent microbial growth).

2. Pre-Use Treatment of Previously Used Electroporation Cuvettes:

a. Water Rinse: Take the dry-stored cuvette from the freezer. Inside the laminar flow hood, add ddH₂O and use a 1 mL pipette to aspirate and dispense twice inside the chamber. Shake dry.
b. Ethanol Rinse: Inject 75% ethanol into the chamber and use a 1 mL pipette to aspirate and dispense twice. Shake dry.
c. UV Sterilization: Arrange the cuvettes upright directly under the UV lamp in the hood. Align the electrode gap parallel to the lamp tube, and orient the inner surface of the cuvette lid towards the UV lamp to ensure direct UV exposure to the interior. UV sterilize for ~20 minutes.
d. Sterile Water Rinse: Rinse the cuvette twice with sterile ddH₂O and shake dry.
e. Pre-cooling: Place the cuvette on ice for pre-cooling. Allow ventilation in the laminar flow hood to further dry the interior of the cuvette before proceeding with the subsequent transformation steps.

## Colony PCR

### Ⅰ. Experimental Materials

**1. Biological Materials**

- Colonies to be tested: Transformed Lacticaseibacillus rhamnosus (Experimental group
- Positive control: Escherichia coli containing the target plasmid
- Negative control: Lacticaseibacillus rhamnosus with empty vector

**2. Reagents**

- PCR Pre-mix: 2× Taq Master Mix (contains dye)
- Primer pairs
- Colony lysis solution: 20 mM NaOH containing 0.5% SDS
- TE Buffer: 10 mM Tris-HCl, 1 mM EDTA
- Agarose gel electrophoresis reagents: 1× TAE Buffer, 1.5% agarose gel (contains GelRed nucleic acid dye), DNA molecular weight standard

**3. Equipment**

- PCR machine, Gel electrophoresis system, Gel imaging system, Constant temperature incubator, Micropipettes and sterile tips

### Ⅱ. Experimental Procedure

**① Experimental Preparation:**

Turn on the metal bath and set it to 95°C. Prepare MRS agar plates with erythromycin resistance, a marker pen, lysis solution, EP tubes, sterilized pipette tips, etc.

**② Colony Preparation (Aseptic Technique):**

Pick single colonies from the transformation plate and pre-culture them at marked positions on a new MRS plate. Partition the bottom of the petri dish with a marker pen, inoculating only one colony type per sector. Strain names can be written on the lid, but sector divisions and numbers must be marked on the bottom of the dish. Use a 10μL pipette tip to pick a single colony, inoculate it onto the correspondingly labeled sector of the new plate to ensure transfer, and then place the tip with the attached colony directly into the lysis solution (the tip can be expelled into an EP tube containing lysis solution). Continue picking the next colony. Mark each tube to correspond with the marking on the new plate.

③ Colony Lysis & Template Preparation

1. Lysis Solution Method:

Add 20 μL of lysis solution (20 mM NaOH + 0.5% SDS) to a PCR tube/EP tube. Gently touch a colony with a sterilized 10μL pipette tip, first inoculate it onto the new plate, then resuspend it in the lysis solution. Heat at 95°C for 10 minutes, immediately place on ice for 2 minutes, add 180 μL of TE Buffer (or ddH₂O) to neutralize, centrifuge at 12,000 × g for 2 minutes, and use the supernatant as the template.

2. Boiling Method:

Suspend the colony in 50 μL of sterile water. Heat at 95°C for 10 minutes, centrifuge at 12,000 × g for 5 minutes, and use the supernatant as the template.

④PCR Reaction System

| Component | Volume |
| --- | --- |
| 2× Taq Master Mix | 10 μL |
| Forward Primer (10 μM) | 1 μL |
| Reverse primer (10 μM) | 1 μL |
| Template DNA | 2 μL |
| ddH₂O | 6 μL |

⑤PCR Reaction Program

| Step | Temperature | Time | Number of Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 95 ℃ | 5 min | 1 |
| Denaturation | 95 ℃ | 30 sec | 30 |
| Annealing | 55 ℃ | 30 sec | — |
| Extension | 72 ℃ | 45 sec/kb | — |
| Terminal extension | 72 ℃ | 5 min | 1 |
| Save | 4 ℃ | ∞ | — |

**Note:** Optimize the annealing temperature according to the primer Tm values.

⑥Electrophoresis Analysis

Prepare a 1% agarose gel containing nucleic acid dye. Load the samples and perform electrophoresis at 120 V. Conduct gel imaging analysis after 25-30 minutes.

#### Plasmid Target Fragment and Primer Information

| Module | Serial Number | Resistance | expression | Plasmid Size | Primer Name | Sequence (5' to 3') |
| --- | --- | --- | --- | --- | --- | --- |
| Sensing Module | 1 | Erythromycin | 8852-eGFP | 352bp | Pnps-F | CGGATTTTACGCCGTGTACTGG |
|  | 2 | Erythromycin | 8852-eGFP | 728bp | medi8852-F | CTGTCTTCCTACACTCACTG |
| Response Module | 3 | Erythromycin | 8852-mCherry | 714bp | 8852-F | CAGGTCTGGTTAAACCGTCTC |
|  | 4 | Erythromycin | 1G01-mCherry | 822bp | 1G01'-F | GTTCAGCTGGTCGAATCTGG |
| Security Module | 5 | Erythromycin | MazF | 618bp | ZT-MazF'-F | TGTCAGATAGGCCAATGACTG |
|  | 6 | Erythromycin | CI-qR-GFP-srrA | 867bp | CI-R | TATATTACAGCTCCAGATCTACCG |
|  | 6 | Erythromycin | YF1-FixJ-FixK2-GFP | 835bp | FixJ-YF1-F | TTTCTTCACCACCAAGGACAC |
|  | 7 | Erythromycin | GFP | 1072bp | ZT-GFP-F | CAACACGTGCTGTAATTTGAAGC |
|  | 8 | Erythromycin | RNAthermo-GFP | 1110bp | ZT-GFP-F | CAACACGTGCTGTAATTTGAAGC |
|  | 9 | Erythromycin | TlpA-pTlpA-GFP | 853bp | ZT-pTlpA-GFP-F | CATAAGGGAGAGCGTCGAGATC |
|  | 10 | Erythromycin | TlpA-pTlpA-GFP | 1456bp | ZT-TlpA-F | CGGCGTAGAGGATCGAGATCT |

## Fluorescent Verification

Plasmid Expression, Secretion Localization, and Viral Response Kinetics in Genetically Engineered Probiotics

### Ⅰ. Experimental Materials

**Strains and Plasmids:**

- Recombinant Lacticaseibacillus rhamnosu, Empty vector control strain

**Virus and Reagents:**

- Inactivated influenza A virus (H1N1-PR8 strain), MRS medium, Erythromycin, PBS buffer, etc.

**Equipment:**

- Blue light gel excision instrument, Fluorescent microplate reader, Constant temperature shaker, Centrifuge, etc.

### Ⅱ. Experimental Procedure

**Bacterial Culture:**

- Inoculate recombinant and control strains into 5 mL of MRS medium containing antibiotics, and culture overnight at 37°C.
- Centrifuge at 5000 × g for 10 min to collect the bacterial cells. Wash the pellet three times with PBS.

**Fluorescence Expression:**

- Inoculate the recombinant bacteria into a 96-well plate with black walls and a clear bottom, using 200μL per well.
- Place the plate under UV or blue light to observe fluorescence plasmid expression either at different time points under the same treatment conditions or under different treatments at the same time point.
- Use ImageJ for fluorescence intensity data analysis: Extracellular fluorescence intensity = Total fluorescence - Intracellular fluorescence.

## Influenza Virus HA Hemagglutination Assay

The hemagglutination inhibition (HI) assay is a traditional serological method used to detect antibodies in serum that can inhibit the hemagglutination activity of influenza A viruses. This assay is based on the property of the influenza A hemagglutinin (HA) to bind to red blood cells, causing agglutination. If antibodies against HA are present in the serum, they will inhibit the viral hemagglutination. By measuring the maximum dilution of serum that inhibits agglutination, the antibody level can be quantified.

After culturing the influenza virus, we typically determine the titer of the cultured virus via the hemagglutination assay. The hemagglutinin (HA) on the surface of the influenza virus can cause agglutination of specific red blood cells, allowing for rapid estimation of the influenza virus titer.

1. Incubate the engineered bacteria and wild-type bacteria with inactivated influenza A virus at 37°C for 2 hours.

2. Take the 1% chicken red blood cell (RBC) suspension, centrifuge at 1000 rpm for 10 min, discard the supernatant, and resuspend/wash the pellet with PBS. Repeat this process three times until hemolyzed cells are removed and the supernatant appears clear after centrifugation.

3. Add 50 μL of PBS to each well of a V-bottom microtiter plate. Add 50 μL of each sample to be tested to the wells of the first row, using three wells per sample type.

4. Using a multichannel pipette, serially dilute the mixture by transferring 50 μL from the wells in the first row to the corresponding wells in the subsequent rows (a 2-fold serial dilution across 8 rows). Discard 50 μL from the final row after mixing, so that all wells contain a final volume of 50 μL.

5. Add 50 μL of the RBC suspension to each well. Let the plate stand at room temperature for 30 minutes and then observe the results.

6. For result interpretation, lift the plate and tilt it at an angle towards the ground. Observe the wells from the back. In wells where agglutination did not occur, the RBCs will stream down in a tear-shaped pattern due to gravity. Record the observations.

**Note:** Retain one row of wells without any added virus sample as a negative control. No agglutination should occur in these control wells.

## ELISA

Enzyme-Linked Immunosorbent Assay

It is used for the quantitative detection of specific antibodies against influenza A virus in serum. The ELISA method offers advantages of high sensitivity and high throughput. By measuring the amount of antibody binding, the strength of the immune response to the virus in the host can be assessed.

1. **Coating:**  inactivated influenza A virus in 1\* PBS to a total volume of 10ml. Add 100μl per well to a 96-well ELISA plate. Incubate overnight at 4°C.

2. **Washing:** Wash the plate 5 times with washing buffer (1\* PBST: 1L PBS + 500μl Tween-20). Tap the plate dry after washing.

3. **Blocking:** Prepare 20ml of 5% skimmed milk. Add 200μl per well. Incubate overnight at 4°C.

4. **Washing:** Wash the plate 5 times with washing buffer (1\* PBST). Tap the plate dry.

5. **Primary Antibody:** Add 100μl of PBS to each well. Add 100μl of engineered bacterial culture and wild-type bacterial culture (with similar OD₆₀₀ readings) to the wells of the first row. Mix well, then transfer 100μl from the first row to the second row. Continue this serial dilution across the plate. Place the entire plate in a sterile 37°C incubator and incubate for 2 hours.

6. **Washing:** Wash the plate 5 times with washing buffer (1\* PBST). Tap the plate dry.

7. **Secondary Antibody:** Add 100μl per well of 5% skimmed milk containing His-tag antibody. Incubate in a sterile 37°C incubator for 1 hour.

8. **Washing:** Wash the plate 5 times with washing buffer (1\* PBST). Tap the plate dry.

9. **Color Development:** Add 100μl of chromogenic substrate solution per well. Develop color in the dark for 15 minutes.

10. **Stop Reaction:** Add 50μl of stop solution per well. Measure the OD₄₅₀.

## Bacterial Cell Membrane Lysis

1. Centrifuge 10ml of bacterial culture, discard the supernatant, and transfer the pellet to a 50ml centrifuge tube.

2. Add 15ml of lysis buffer and resuspend the pellet thoroughly using a pipette.

3. Secure the centrifuge tube in a beaker filled with ice and a small amount of ice water. Set the ultrasonic cell disruptor parameters: Power 30%, Total Time 30 minutes, Pulse: ON for 2s, OFF for 8s. Select a 6mm ultrasonic probe tip and immerse it approximately 2cm below the liquid surface at the center. Begin cell lysis.

4. After lysis, wipe the probe with 75% ethanol and rinse with ddH₂O, then dry.

5. Centrifuge the lysate at 4000 rpm for 5 minutes. Collect the supernatant.

## GFP Expression

Regulated Expression of Heat-Induced TlpA

**1. Picking Colonies:**

Pick colonies from a solid agar plate, categorizing them into experimental group, negative control group, and positive control group. Inoculate the picked colonies into 5 ml of MRS medium containing erythromycin. Culture overnight at 30°C.

**2. Inoculation and Divided Induction:**

Take the overnight culture and inoculate 1% of it into 5 ml of MRS medium containing erythromycin. Incubate statically at 30°C for 10 hours.

**3. Fluorescence Intensity and OD₆₀₀ Measurement:**

Divide the culture for induction at different temperatures (30°C, 37°C, 45°C). Simultaneously, prepare a 96-well plate with 190μl PBS per well. After 3 hours of induction, add 10μl of each sample to the wells. Measure fluorescence intensity and OD₆₀₀ using a microplate reader. Use MRS medium as a negative control.

**4. Data Analysis:**

Use the formula Fl/OD = [Fl(sample) - Fl(blank)] / [OD₆₀₀(sample) - OD₆₀₀(blank)] to correct for background fluorescence. Repeat the experiment three times, calculate the average, and remove outliers.

## GFP Expression

Regulated Expression of Heat-Induced RNA (ROSE)

**1. Picking Colonies**

Pick colonies from the solid agar plate, categorizing them into experimental group, negative control group, and positive control group. Inoculate the picked colonies into 5 ml of MRS medium containing erythromycin. Culture overnight at 28°C.

**2. Inoculation and Divided Induction**

Take the overnight culture and inoculate 1% of it into 5 ml of MRS medium containing erythromycin. Incubate statically at 28°C for 12 hours.

**3. Fluorescence Intensity and OD₆₀₀ Measurement**

Divide the culture for induction at different temperatures (28°C, 30°C, 37°C). Simultaneously, prepare a 96-well plate with 190μl PBS per well. At time points 0.5h, 1h, 3h, and 5h after induction, add 10μl of each sample to the wells. Measure the fluorescence intensity and OD₆₀₀ values using a microplate reader. Use MRS medium as a negative control.

**4. Data Analysis**

Use the formula Fl/OD = [Fl(sample) - Fl(blank)] / [OD₆₀₀(sample) - OD₆₀₀(blank)] to correct for background fluorescence. Repeat the experiment three times, calculate the average, and remove outliers.

## Promoter Expression

Small Molecule Peptide (IF) Induction Experiment

1. Pick a positive transformation clone and inoculate it into 5 ml of MRS broth medium containing erythromycin. Culture overnight at 37°C.

2. Dilute the culture 1:100 into 50 ml of MRS broth medium containing erythromycin. Culture at 37°C for 8 hours.

3. Add synthetic IF-673, setting up concentration gradients: 0 ng/ml (control), 25 ng/ml, 50 ng/ml, 100 ng/ml. Continue incubation at 37°C for 30 minutes. Select the appropriate concentration gradient for subsequent induction experiments.

## Blue Light and Natural Light Induction

Functional Verification of YF1-FixJ-FixK2-GFP

**(YF1-FixJ-FixK2-GFP)**

**1. Picking Colonies**

Pick colonies from the solid agar plate, categorizing them into experimental group, negative control group, and positive control group. Inoculate the picked colonies into 5 ml of MRS medium containing erythromycin. Culture overnight at 37°C.

**2. Inoculation and Divided Induction**

Take the overnight culture and inoculate 1% of it into MRS medium containing erythromycin. Culture statically in darkness for 8 hours.

**3. Fluorescence Intensity and OD₆₀₀ Measurement**

Monitor the change in OD₆₀₀ value and measure the fluorescence intensity / OD₆₀₀ value using a microplate reader.

Prepare a 96-well plate, adding 190μl PBS to each well. 3 hours later, add 10μl of sample to each well. Measure the fluorescence intensity / OD₆₀₀ value using the microplate reader, using MRS medium as a negative control.

**4. Data Analysis**

Use the formula Fl/OD = [Fl(sample) - Fl(blank)] / [OD₆₀₀(sample) - OD₆₀₀(blank)] to correct for background fluorescence. Repeat the experiment three times, calculate the average, and remove outliers.

## Repressor Protein Induction

Functional Verification of the CI-pR-GFP

**1. Picking Colonies**

Pick colonies from the solid agar plate, categorizing them into experimental group, negative control group, and positive control group. Inoculate the picked colonies into 5 ml of MRS medium containing erythromycin. Culture overnight at 37°C.

**2. Inoculation and Divided Induction**

Take the overnight culture and inoculate 1% of it into MRS medium containing erythromycin. After static culture for 8 hours, add the IF-673 inducer.

**3. Fluorescence Intensity and OD₆₀₀ Measurement**

Closely monitor the change in OD₆₀₀ value and measure the fluorescence intensity / OD₆₀₀ value using a microplate reader.

Prepare a 96-well plate, adding 190μl PBS to each well. 3 hours later, add 10μl of sample to each well, and measure the fluorescence intensity / OD₆₀₀ value using the microplate reader, using MRS medium as a negative control.

**4. Data Analysis**

Use the formula Fl/OD = [Fl(sample) - Fl(blank)] / [OD₆₀₀(sample) - OD₆₀₀(blank)] to correct for background fluorescence. Repeat the experiment three times, calculate the average, and remove outliers.

- [*![](https://static.igem.wiki/teams/5840/figure/background.webp)
  LGG GROWTH CURVE*](#LGG Growth Curve)

- [*![](https://static.igem.wiki/teams/5840/figure/ret-3.webp)
  PLASMID TARGET
             FRAGMENT AND
             PRIMERINFORMATION*](#Plasmid Target Fragment and Primer Information)

- [*![](https://static.igem.wiki/teams/5840/figure/ret-1.webp)
  RESPONSE MODULE*](#Response Module)

- [*![](https://static.igem.wiki/teams/5840/figure/inspiration.webp)
  ELISA*](#Elisa)

- [*![](https://static.igem.wiki/teams/5840/figure/ret-2.webp)
  FUNCTION PLASMID
             EXPRESSION IN BACTERIA*](#Functional Plasmid Expression in Engineered Bacteria)

- [*![](https://static.igem.wiki/teams/5840/figure/ret-4.webp)
  SAFETY MODULE*](#Safety Module)

## LGG Growth Curve

### Growth Curve of Wild-Type Lactobacillus casei rhamnosus

![](https://static.igem.wiki/teams/5840/figure/result-p1.webp)

The experimental results of the growth curve determination of wild-type Lactobacillus casei rhamnosus show that after 24 hours of incubation at 37°C in a shaking incubator and under static culture conditions, the bacterial growth curves are generally similar. During the lag phase (0–3 hours), the bacteria adapt to the new environment and prepare for division, with the population remaining essentially unchanged. During the logarithmic phase (4–16 hours), the bacteria divide rapidly in an exponential manner, resulting in a sharp rise in the curve. In the stationary phase (17–24 hours), nutrient depletion and metabolite accumulation lead to a balance between the number of new and dead bacterial cells, which is consistent with expectations.

### Growth Curve of Recombinant Lactobacillus casei rhamnosus under Antibiotic Resistance Conditions

![](https://static.igem.wiki/teams/5840/figure/en-88.webp)

The experimental results of the growth curve determination of recombinant Lactobacillus casei rhamnosus under antibiotic resistance conditions show a deviation from the trend observed in the wild-type strain. Specifically, the lag phase of the recombinant strain is significantly prolonged under resistance conditions, likely due to the inhibition of bacterial proliferation by erythromycin during this phase. As a result, the time required for the bacterial population to reach the mid-logarithmic phase is extended. Nevertheless, the overall trend still conforms to the general pattern of bacterial growth curves.

### Growth Curve of Recombinant Lactobacillus casei rhamnosus under Non-Resistance Conditions

![](https://static.igem.wiki/teams/5840/figure/result-p22.webp)

The experimental results of the growth curve determination of recombinant Lactobacillus casei rhamnosus under non-resistance conditions indicate that the trend is generally consistent with that of the wild-type strain, which aligns with expectations.

## Plasmid Target Fragment and Primer Information

| Module | Serial Number | Resistance | expression | Plasmid Size | Primer Name | Sequence (5' to 3') |
| --- | --- | --- | --- | --- | --- | --- |
| Sensing Module | 1 | Erythromycin | 8852-eGFP | 352bp | Pnps-F | CGGATTTTACGCCGTGTACTGG |
|  | 2 | Erythromycin | 8852-eGFP | 728bp | medi8852-F | CTGTCTTCCTACACTCACTG |
| Response Module | 3 | Erythromycin | 8852-mCherry | 714bp | 8852-F | CAGGTCTGGTTAAACCGTCTC CTTTGATTTCTACCTTGGTGCC CACGTGCTGTAATTTGAAGC CTGTCGAAGTATTGCTGGTAC |
|  | 4 | Erythromycin | 1G01-mCherry | 822bp 839bp | 1G01'-F 1G01'-R ZT-pho-F pho-R | GTTCAGCTGGTCGAATCTGG GATGGCCATGTTATCCTCCTC CACGTGCTGTAATTTGAAGC CTGTCGAAGTATTGCTGGTAC |
| Security Module | 5 | Erythromycin | MazF | 618bp | ZT-MazF'-F MazF'-R | TGTCAGATAGGCCAATGACTG CCAATCAGTACGTAAAATTTGGC |
|  | 6 | Erythromycin | CI-qR-GFP-srrA | 867bp | CI-R ZT-CI-F | TATATTACAGCTCCAGATCTACCG CACTGACTAGCGATAACTTTCC |
|  | 6 | Erythromycin | YF1-FixJ-FixK2-GFP | 835bp | FixJ-YF1-F ZT-FixJ-YF1-R | TTTCTTCACCACCAAGGACAC GGTCGACAATGAGTGAGCTAAC |
|  | 7 | Erythromycin | GFP | 1072bp | ZT-GFP-F GFP-R | CAACACGTGCTGTAATTTGAAGC CACTTGTACAGCTCGTCCATG |
|  | 8 | Erythromycin | RNAthermo-GFP | 1110bp | ZT-GFP-F GFP-R | CAACACGTGCTGTAATTTGAAGC CACTTGTACAGCTCGTCCATG |
|  | 9 | Erythromycin | TlpA-pTlpA-GFP | 853bp | ZT-pTlpA-GFP-F GFP-R | CATAAGGGAGAGCGTCGAGATC CACTTGTACAGCTCGTCCATG |
|  | 10 | Erythromycin | TlpA-pTlpA-GFP | 1456bp | ZT-TlpA-F tlpA-R | CGGCGTAGAGGATCGAGATCT   CTGGCCACCGGTCTGTTTATTG |

## Sensing Module

### Colony PCR

![](https://static.igem.wiki/teams/5840/figure/en-6.webp)

![](https://static.igem.wiki/teams/5840/figure/en-98.webp)

![](https://static.igem.wiki/teams/5840/figure/en-57.webp)

Single colonies of recombinant bacteria after electrotransformation, selected via antibiotic screening, were picked from MRS agar plates containing erythromycin resistance for colony PCR. The picked colonies were retained for subsequent fluorescent protein expression verification. The video shows the sensing module recombinant Lacticaseibacillus rhamnosus selected through antibiotic screening, where the colonies appear smooth and off-white.

The image shows the positive PCR results of the plasmid primers for the engineered bacterial sensing module, with bands at the correct positions. The experiment preliminarily confirms that the designed plasmid of the sensing module has been successfully transferred into Lacticaseibacillus rhamnosus.

### Response of the Sensing Module Under Viral Gradient Stimulation

![](https://static.igem.wiki/teams/5840/figure/en-89.webp)

fig.4

The inactivated influenza A virus (H1N1-PR8 strain) measured in chicken red blood cells had a HAI= 1024(Figure 4).

![](https://static.igem.wiki/teams/5840/figure/en-90.webp)

fig.5

Group 1: Engineered bacteria without inactivated influenza A virus.

Group 2: Engineered bacteria with 10^1-fold diluted inactivated influenza A virus.

Group 3: Engineered bacteria with 10^2-fold diluted inactivated influenza A virus.

Group 4: Engineered bacteria with 10^3-fold diluted inactivated influenza A virus.

Group 5: Engineered bacteria with 10^4-fold diluted inactivated influenza A virus.

Group 6: Engineered bacteria with 10^5-fold diluted inactivated influenza A virus.

Group 7: Engineered bacteria with 10^6-fold diluted inactivated influenza A virus.

The virus was diluted gradiently to stimulate the recombinant bacteria, and the expression of fluorescent proteins was monitored using a microplate reader at specific wavelengths(Figure 5). Experimental data show that as the viral dilution gradient increased, the fluorescent protein expression of the recombinant bacteria gradually increased until the dilution factor reached 10⁷, where it leveled off compared to the control group. This suggests that the growth and viability of the recombinant bacteria were inhibited at high viral titers, while at low to medium viral loads, they sensed the presence of the virus and responded.

## Response Module

### Co-transformation of the Sensing and Response Module Plasmids

![](https://static.igem.wiki/teams/5840/figure/re-23.webp)

fig.1

![](https://static.igem.wiki/teams/5840/figure/re-24.webp)

fig.2

Preliminary experiments were conducted using E. coli containing the respective module plasmids. The E. coli cells containing the functional sensing module plasmid were prepared into competent cells, which were then transformed with the response module plasmid and incubated. The successful electroporation parameters are shown above.

### Colony PCR

![](https://static.igem.wiki/teams/5840/figure/en-91.webp)

fig.4

Colonies returned two diagnostic bands corresponding to both the sensing plasmid and the newly introduced response plasmid. PCR validation showed correctly sized bands in the second batch of engineered bacteria.

These results indicate that both the sensing module and response module plasmids were successfully transformed into the engineered bacteria.

### Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

![](https://static.igem.wiki/teams/5840/homenew/tiaodai.avif)

fig.3

SDS-PAGE of bacterial lysates indicated scFv protein expression, though mixed with faint non-specific signals.

### Hemagglutination Inhibition Assays

![](https://static.igem.wiki/teams/5840/figure/en-25.webp)

fig.1

![](https://static.igem.wiki/teams/5840/figure/en-26.webp)

fig.2

![](https://static.igem.wiki/teams/5840/figure/en-92.webp)

fig.3

Figure 1 8852mc group hemoconcentration level: +; 1G01 group hemoconcentration level: ++

Figure 2 Virus group hemoconcentration level: ++++

Figure 3 WT group hemoconcentration level: +++

Supernatant from induced dual-plasmid strains tested by **HA inhibition assay** shows the reduction in hemagglutination, indicating successful secretion and neutralizing activity.

## Elisa

![](https://static.igem.wiki/teams/5840/figure/re-39.webp)

fig.4

Based on the ELISA indirect assay, we observed that the engineered bacterial group exhibited lower levels of unbound anti-His tag mouse mAbs compared to the wild-type bacterial group. Furthermore, the upward trend in antibody levels was more pronounced during serial dilutions. This indicated the presence of scFv antibodies within the total protein extracted from the engineered bacteria.

###

## Functional Plasmid Expression in Engineered Bacteria

### Fluorescence Verification

![](https://static.igem.wiki/teams/5840/figure/result-p17.webp)

fig.1
Sensing Module

![](https://static.igem.wiki/teams/5840/figure/result-p15.webp)

fig.2
Response Module

![](https://static.igem.wiki/teams/5840/figure/result-p16.webp)

fig.3
Sensing and Response Module

![](https://static.igem.wiki/teams/5840/figure/result-p18.webp)

fig.4
Safety Module

![](https://static.igem.wiki/teams/5840/figure/result-p19.webp)

fig.5
Safety Module

![](https://static.igem.wiki/teams/5840/figure/result-p20.webp)

fig.6
Safety Module

Figure 1 shows the expression of the eGFP green fluorescent plasmid in colonies of the sensing module Lactobacillus casei rhamnosus.

Figure 2 shows the expression of the mCHERRY red fluorescent plasmid in colonies of the response module Lactobacillus casei rhamnosus.

Figure 3 shows the co-expression of the red and green fluorescent plasmids in colonies after co-transformation with both the sensing and response module plasmids.

Figures 4-6 show the expression of the eGFP green fluorescent plasmid in colonies of the safety module Lactobacillus casei rhamnosus.

All colonies retained were PCR-positive with bands at the correct positions. The three groups on each plate represent three different single colonies from the same antibiotic plate and the three adjacent colony spots originate from the same single colony picked via antibiotic screening, representing three biological replicates.

All plates were MRS agar plates with erythromycin resistance. Observations indicate that the fluorescent plasmids designed for all the modules were successfully expressed.

## Safety Module

### Colony PCR

![](https://static.igem.wiki/teams/5840/figure/en-93.webp)

fig.1
colony PCR for TlpA-pTlpA-GFP

![](https://static.igem.wiki/teams/5840/figure/en-94.webp)

fig.2
Colony PCR for RNAthermo & eGFP

![](https://static.igem.wiki/teams/5840/figure/en-95.webp)

fig.3
colony PCR for CI-pR

![](https://static.igem.wiki/teams/5840/figure/en-96.webp)

fig.4
colony PCR for YF1-FixJ-FixK2-GFP

![](https://static.igem.wiki/teams/5840/figure/en-97.webp)

fig.5
colony PCR for MazF

PCR products matched the theoretical sizes predicted from primer design, confirming successful amplification of the target fragments

### Temperature-induced eGFP expression

![](https://static.igem.wiki/teams/5840/figure/re-32.webp)

fig.1
TlpA system validation

![](https://static.igem.wiki/teams/5840/figure/re-33.webp)

fig.2
RNA thermometer(ROSE) validation experiment

![](https://static.igem.wiki/teams/5840/figure/re-34.webp)

![](https://static.igem.wiki/teams/5840/figure/re-35.webp)

fig.3
Secondary-structure prediction of RNA thermometers

1. TlpA system: TlpA switch ON >37 °C in 3h as expected (Fig 1).

2. RNA thermometer: dip 25–30 °C, peak 37 °C, opposite to ROSE rule; modeling shows identical long-stem loops 25–45 °C, melt only at 50 °C (Fig 2-3). Now, the modeling team has abandoned single-shot static predictions and is now collaborating with the experimental group to build a four-dimensional dataset of temperature–time–fluorescence–mRNA half-life

### Blue light kill switch assay

![](https://static.igem.wiki/teams/5840/figure/en-52.webp)

fig.4
pdawn elements validation

![](https://static.igem.wiki/teams/5840/figure/re-37.webp)

fig.5
CI-PR Induction Assay

![](https://static.igem.wiki/teams/5840/figure/re-38.webp)

fig.6
MazF toxin validation

1. Fluorescence detection: As anticipated, under dark conditions, the FixK2 promoter was activated, producing eGFP and yielding a markedly stronger fluorescence signal. Following 2 hours of blue light exposure, a pronounced suppression of fluorescence was observed, confirming that blue light successfully inhibited FixJ-YF1, thereby suppressing the expression of the eGFP fluorescent protein (Fig.4). Under 75 ng/mL IP-673 induction, the CI-PR system functions normally: CI represses eGFP expression downstream of PR.

2. Serial-dilution spread-plate assay: Induction with 7 ng/mL IP-673 triggered MazF expression and confirmed its toxic effect for at least 9h (fig.6).