iGEM 2025 — Team ehsbnu

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{% block title %}Experiment Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
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# EXPERIMENT NOTEBOOK

![DNA Divider]({{ url_for('static', filename='cache/DNAline.png') }})

Experiment Phases

* [Bacmid Transformation](#bacmid-transformation)
* [Virus Transfection](#virus-transfection)
* [Cell Lab](#cell-lab)

## ![Bacmid Icon]({{ url_for('static', filename='cache/logo.png') }}) Bacmid Transformation and Bacterial Culture

📅 8.18

Operator: Miao Lexing, Zhang Yichi

* Prepared 200ml LB medium and sterilized it
* Diluted CM plasmid to 400 ng/μl, added 1μl to competent cells, and let stand for 30 minutes
* Heat shock at 42°C for 90 seconds, added 1ml antibiotic-free LB medium for recovery (4 hours, 37°C shaker at 180 rpm)
* Stored remaining plasmid at -20°C
* Prepared culture plates: 200ml LB medium, added 200μl Kanamycin, Gentamicin, Tetracycline, and Bluo-gal, added 33μl IPTG. Poured into ten culture dishes
* Diluted one group of bacterial solution to 10x and 100x. Took 200μl each of 1x, 10x, and 100x bacterial solutions and spread evenly onto triple-antibiotic plates. Cultured in shaker for two days

📅 8.25

Operators: Yang Ruiming, Miao Lexing

* Checked culture results from 8.18, selected two larger white colonies, and froze them
* Performed transformation on the remaining three plasmids (MS, SC, MCS): Diluted each to 400ng/μl, added 1μl to competent cells, stood for 30min
* Heat shock at 42°C for 90 seconds, added 1ml antibiotic-free LB medium for recovery (4 hours, 37°C shaker at 180 rpm)
* Stored remaining plasmids at -20°C
* For the three groups of bacterial solutions, diluted 200μl to 10x for each. Spread both original and diluted solutions onto triple-antibiotic plates. Cultured for two days

📅 8.28

Operators: Zhang Jingtian, Miao Lexing

* The 8.25 cultures showed no growth, suspected issue with competent cells
* Repeated transformation with new competent cells, operation identical to 8.25
* Prepared 200ml triple-antibiotic LB during recovery period

📅 8.31

Operators: Miao Lexing, Zhang Jingtian, Yang Ruiming

* Prepared 4 groups: 6ml medium \* 4, with antibiotics (Kanamycin, Tetracycline, Gentamicin (1000x))
* Picked 2 SC white colonies and 2 CM white colonies
* Shaker culture for 1 day

📅 9.1

Operators: Miao Lexing, Zhang Jingtian, Yang Ruiming

* Repeated transformation of SM and MCS plasmids, operation same as above
* Performed viral plasmid extraction
* Measured concentration, sufficient for next step experiments

## ![Virus Icon]({{ url_for('static', filename='cache/logo.png') }}) Virus Transfection and Cell Culture

📅 9.14

Electrophoresis

* Order from left to the right: SC1 SC2 MS1 marker MS2 CM1 CM2

📅 9.15

Operator: Yang Ruiming

* Took 14 tubes, added 100μl insect cell medium to each
* Added 10μl transfection reagent to 7 tubes, mixed
* Added virus plasmid (1μg each) to the other 7 tubes, mixed gently by flicking
* Added 200μl medium containing transfection reagent to these 7 tubes
* Took the 6-well plate from the previous day, removed supernatant, added 800μl medium containing the plasmid mixture, mixed, and added to the 6-well plate
* Cultured for 5 days

📅 9.20

Virus Harvesting

* Harvested supernatant from the 6-well plate cultured on the 15th
* Took two new 6-well plates, added 0.5mL cell culture medium (containing cells) to each well
* Prepared three culture dishes, added 2mL of remaining cells to each dish
* Added 8mL medium to each culture dish
* Added all harvested virus to the aforementioned 6-well plates, with virus type corresponding to each plate
* Cultured at 28°C for 3 days

📅 9.27

Western Blotting - Primary Antibody

* Suspended cells in 1mL buffer
* Added 20mL protease inhibitor, then lysed with ultrasound for 30s
* Mixed 80μl protein + 20μl dye, metal bath at 95°C for 10 min
* Loaded gel (left to right): Marker (5μl) + seven samples (10μl each) + Marker (2.5μl). Electrophoresis at 120V for 50 min, transfer at 400mA for 2h
* Added skim milk for blocking, 1h
* Added skim milk containing primary antibody, placed in 4°C refrigerator for 12-16 hours

## ![Cell Lab Icon]({{ url_for('static', filename='cache/logo.png') }}) Cell Lab

Key Activities involving Cells

Virus transfection and amplification in insect cells (9.15, 9.20)

Cell collection and lysis for Western Blotting (9.27)

Cell Freezing & Viability Assessment: Preparation of freezing medium, cell detachment, dividing into replicate tubes, and Trypan Blue staining to record viability for the purpose of comparing cell viability before and after the freeze-thaw process (9.29)

Cell Thawing & Viability Assessment: Thawing of cells, warm water renaturation, and statistical viability assessment for the purpose of comparing cell viability before and after the freeze-thaw process (9.30)

Subsequent culture of thawed cells (10.2)

📅 9.29

Operators: Yang Ruiming, Zhang Jingtian, Miao Lexing

* Prepared cell freezing medium
* Detached cells, divided cells corresponding to each plasmid into three tubes as replicates
* Performed Trypan Blue staining to record viability for the purpose of comparing cell viability before and after the freeze-thaw process

📅 9.30

Western Blotting - Secondary Antibody

* Membrane regeneration solution, shaker for 15 min
* Prepared 50ml 5% skim milk
* Took cells, warm water renaturation, statistically viability for the purpose of comparing cell viability before and after the freeze-thaw process

📅 10.2

First Experiment Cycle Completion

* The first experiment cycle completed
* Transferred the cells resuscitated the previous day to culture dishes, added 5ml medium, and continued cultivation

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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# ENGINEERING - BACMID DESIGN

![DNA Divider]({{ url_for('static', filename='cache/DNAline.png') }})

## Bacmid Design Strategy

We have constructed four specific bacmids (M, C, and S refer to containing MAHS, CAHS, and SAHS genes in the bacmids, respectively): the experimental design containing all three target proteins (MCS) and three negative controls (CM, MS, SC).

## Expression System Design

Key Design Elements

Polyhedrin Promoter: A strong promoter commonly used in baculovirus systems for efficient expression

P2A Peptide Sequences: Enable ribosomal skipping during translation, ensuring production of individual proteins from a single transcript

Tandem Protein Arrangement: Three proteins arranged sequentially for coordinated expression

The three proteins were arranged in tandem and separated by P2A peptide sequences, which induced ribosomal skipping during translation and thereby ensured production of individual proteins from a single transcript.

## Regulatory System Design

To further refine the system, we incorporated OsTIR1 as a regulatory element to facilitate protein degradation and promote cellular recovery after expression.

Auxin-Inducible Degradation System

To enable auxin-induced degradation, we fused an AID (Auxin-Inducible Degron) tag to the end of each protein sequence, positioned immediately upstream of the corresponding P2A site. This design allowed us to selectively degrade the expressed proteins by adding auxin after thawing, thereby accelerating cell recovery and improving viability.

## Engineering Design Cycle

Design → Build → Test → Learn

Design: Comprehensive bacmid design with regulatory controls

Build: Construction of four specific bacmid variants

Test: Protein expression and cell survival assessment

Learn: Optimization based on experimental results

![Bacmid Design]({{ url_for('static', filename='cache/desc1.jpg') }})

Bacmid negative control SC (SAHS+CAHS)

![Expression System]({{ url_for('static', filename='cache/desc2.jpg') }})

Bacmid negative control SM (SAHS+MAHS)

![Regulatory System]({{ url_for('static', filename='cache/desc3.jpg') }})

Bacmid negative control CM (CAHS+MAHS)

![Experimental Design]({{ url_for('static', filename='cache/desc4.jpg') }})

Bacmid experiment group MCS (MAHS+CAHS+SAHS)

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
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# EXPERIMENTS

![DNA Divider]({{ url_for('static', filename='cache/DNAline.png') }})

## Materials

The experiments were primarily conducted using Sf9 insect cells, E. coli for plasmid propagation, and baculoviruses for gene expression.

Fall Armyworm Cells (Sf9 / Sf21)

The fall armyworm (*Spodoptera frugiperda*) provides two commonly used cell lines: Sf21 and its clonal derivative Sf9. Both originate from ovarian tissue and are valuable in studying insect virology, protein expression, and host–pathogen interactions.

Sf9 cells, with their uniform morphology and suspension growth, are ideal for large-scale recombinant protein production using the Baculovirus Expression Vector System (BEVS). They are cultured at 27–28 °C without CO₂ supplementation.

E. coli

*Escherichia coli* serves as a prokaryotic host for plasmid cloning and manipulation of baculovirus genomes. Using engineered strains such as DH10Bac, baculovirus genomes can be propagated as bacmids—large plasmid constructs modified for genetic engineering before transfection into insect cells.

Baculoviruses

Baculoviruses are large double-stranded DNA viruses infecting insects of the order Lepidoptera. They are non-pathogenic to humans and have been developed as safe biotechnological tools. The BEVS utilizes baculoviruses to express recombinant proteins in insect cells, producing properly folded and post-translationally modified eukaryotic proteins.

Engineered baculoviruses created via E. coli bacmid technology enable precise insertion of foreign genes, facilitating the production of recombinant proteins or virus-like particles.

## Description of Methods

Step 1: Gene Insertion

We inserted MAHS, CAHS, and SAHS genes into the pET-28a plasmid in *E. coli* strain DH10Bac. Three additional combinations were designed as negative controls. Four plasmids were ordered from GenScript for further use.

| Plasmid | MAHS | CAHS | SAHS |
| --- | --- | --- | --- |
| MCS | Yes | Yes | Yes |
| CM | Yes | Yes | No |
| SM | Yes | No | Yes |
| SC | No | Yes | Yes |

Step 2: Bacmid Construction

Recombinant bacmids were generated using the Bac-to-Bac system (Thermo Fisher) with minor modifications. Donor plasmids were transformed into competent DH10Bac cells and plated on LB agar containing selective antibiotics and indicator compounds.

Bacmid DNA was extracted using a large-construct purification protocol involving lysis, neutralization, and isopropanol precipitation. DNA pellets were air-dried and dissolved in water for concentration measurement using a Nanodrop spectrophotometer. Successful bacmid production was verified by gel electrophoresis.

Step 3: Transfection

Fourteen microcentrifuge tubes were prepared with insect cell medium. Transfection reagent was added to half of the tubes, and bacmid DNA (1 µg per tube) to the other half. The mixtures were combined and transferred into six-well plates containing Sf9 cells.

Cells were incubated at 27 °C for five days, with infection and protein expression monitored throughout.

Step 4: Infection and Protein Expression

Sf9 cells were infected with recombinant baculoviruses and incubated at 27 °C. Cells and supernatants were harvested 48 hours post-infection. Recombinant protein expression was analyzed by SDS–PAGE and Western Blot.

Samples were lysed with protease inhibitors and ultrasonication. Protein samples were separated via SDS–PAGE and stained with Coomassie Brilliant Blue. For Western Blot, proteins were transferred, blocked with nonfat milk, and probed with primary and secondary antibodies. Visualization confirmed the expression of target proteins.

![sf9 cells under microscope 1]({{ url_for('static', filename='cache/exp3.jpg') }})

*sf9* cells under microscope

![sf9 cells under microscope 2]({{ url_for('static', filename='cache/exp4.jpg') }})

*sf9* cells under microscope

![samples' SDS-Page]({{ url_for('static', filename='cache/exp5.jpg') }})

samples' SDS-Page

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance.{% endblock %}
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# RESULTS

PCR Proof of Concept • Western Blot Refinement • Pre-commercial Assessment

![DNA Divider]({{ url_for('static', filename='cache/DNAline.png') }})

## Experimental Progress Overview

During our project, we progressed through Bacmid design, amplification, and transfection. Preliminary PCR checks provided initial evidence that the Bacmid successfully entered the baculovirus system, suggesting that our genetic design had the potential to reach host cells as intended.

While these early results are promising, we continued to validate the expression more rigorously through additional analytical methods.

## Protein Expression Validation

As we conducted Western blotting to confirm gene expression, we obtained bands with limited clarity, which we attributed to procedural aspects that affected protein sample quality.

Systematic Process Improvement

We spent time refining the procedure and prepared protein samples again, demonstrating our commitment to methodological rigor and systematic troubleshooting.

## Phenotypic Testing & Genetic Stability Assessment

We attempted phenotypic testing by measuring the overnight survival rate of frozen cells using hemocytometer counts. Analysis revealed that cells undergoing multiple passages showed variable performance.

Careful investigation identified that these cells risked the loss of inserted DNA—a finding that highlights the importance of genetic stability considerations for long-term cell culture applications.

## Technical Validation & Key Insights

Proof of Concept Established: Preliminary PCR indicates successful Bacmid integration, while phenotypic testing confirms the potential of tardigrade proteins for cell protection during storage.

Methodological Refinement Pathway: Western blotting identified procedural optimization opportunities, leading to immediate protocol improvements.

Genetic Stability Challenge Identified: Multiple passages revealed insert retention challenges, informing our risk assessment for commercialization.

Commercial Viability Demonstrated: The core concept of using tardigrade protective proteins shows promise, though further refinement is needed before market readiness.

## Optimization Strategy & Commercial Pathway

Rather than viewing these challenges as setbacks, we treat them as key lessons in the engineering cycle. Based on existing achievements, we have planned our next steps with commercial success in mind.

Strategic Development Pathway

Further investigation and targeted improvement of the transfection step to address DNA retention challenges

Continued refinement of protein analysis methodologies based on lessons learned

Systematic approach to bridge the gap between proof-of-concept and commercial readiness

This optimization pathway is crucial for our product to be successful on the market

## Entrepreneurial Perspective

From an entrepreneurial perspective, these experiences are valuable—they highlight real-world bottlenecks such as unexpected procedural flaws and inconsistent gene expressions that must be addressed for successful commercialization.

By documenting these experiences transparently, we are not only refining our own process but also providing guidance for future teams who may want to scale tardigrade-protein-based solutions.

## Future Outlook

We are excited to continue refining our workflow and anticipate that the next round of experiments will provide clearer outcomes.

These ongoing efforts illustrate the potential of tardigrade proteins to improve cell survival and reduce research costs, demonstrating both scientific curiosity and practical foresight for commercial applications.

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