iGEM 2025 — Team shsbnu-china

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{% block title %}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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# notebook（Chinese Version）

# notebook（English Version）

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{% block title %}Engineering{% endblock %}
{% block lead %}Development and optimization of multifunctional fusion proteins with hydrogel-forming and adhesive properties, tailored for extreme space environments.{% endblock %}
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# Engineering

## Contents

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* [Overview](#header1)
* [Cycle 1: Design and Expression](#header2)
* [Cycle 2: Expression Optimization](#header3)
* [Cycle 3: Functions and Performance](#header4)
* [Cycle 4: Structure Engineering](#header5)
* [Future Plans](#header6)

## Overview

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Space exploration demands materials that can withstand extreme conditions
while remaining lightweight, strong, and adaptable.
Current options lack self-assembling, adhesive, and hydrogel-forming capabilities.
To address this gap, we engineered a fusion protein combining
hagfish intermediate filament proteins
with mussel foot proteins.

The fusion protein can be effectively heterogeneously produced
in E. coli BL21 (DE3)
under the control of the conventional T7-LacO operon.
This biomaterial forms threads and hydrogels with high water content,
strong adhesion, and mechanical resilience.

We assessed the protein yield rate, and tested its hydrogel formation,
adhesive strength, and structural stability, demonstrating its potential as a novel,
multifunctional material for future space applications.

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## Cycle 1: Design and Expression of Hagfish Intermediate Filament and Mussel Foot Fusion Protein

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## Cycle 2: Expression Optimization of Hagfish Intermediate Filament and Mussel Foot Fusion Protein

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## Cycle 3: Functions and Performance of Hagfish Intermediate Filament and Mussel Foot Fusion Protein

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## Cycle 4: Unstructured Domain Removal from rHIF Proteins

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## Future Plans

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## References

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* 1. Koch, E. A.; Spitzer, R. H.; Pithawalla, R. B.; Castillos, F. A.; Parry, D. A. D. Hagfish Biopolymer: A Type I/Type II Homologue of Epidermal Keratin Intermediate Filaments. Int. J. Biol. Macromol. 1995, 17 (5), 283–292. https://doi.org/10.1016/0141-8130(95)98156-S.
* 2. Böni, L. J.; Zurflüh, R.; Widmer, M.; Fischer, P.; Windhab, E. J.; Rühs, P. A.; Kuster, S. Hagfish Slime Exudate Stabilization and Its Effect on Slime Formation and Functionality. Biology Open 2017, 6 (8), 1115–1122.
* 3. Oliveira, P. E.; Chen, D.; Bell, B. E.; Harris, T. I.; Walker, C.; Zhang, H.; Grob, B.; Lewis, R. V.; Jones, J. A. The Next Generation of Protein Super-Fibres: Robust Recombinant Production and Recovery of Hagfish Intermediate Filament Proteins with Fibre Spinning and Mechanical–Structural Characterizations. Microb. Biotechnol. 2021, 14 (5), 1976–1989.
* 4. Sun, R.; Zheng, R.; Zhu, W.; Zhou, X.; Liu, L.; Cao, H. Directed Self-Assembly of Heterologously Expressed Hagfish EsTKα and EsTKγ for Functional Hydrogel. Front. Bioeng. Biotechnol. 2022, 10, 960586.
* 5. Fudge, D. S.; Gardner, K. H.; Forsyth, V. T.; Riekel, C.; Gosline, J. M. The Mechanical Properties of Hydrated Intermediate Filaments: Insights from Hagfish Slime Threads. Biophys. J. 2003, 85 (3), 2015–2027.
* 6. Xue, R.; Wang, J.; Huang, W.; Qiu, Y.; Xu, H.; Wang, R.; Li, S. Efficient Production of Recombinant Hybrid Mussel Proteins with Improved Adhesion. Int. J. Biol. Macromol. 2025, 289, 139937.
* 7. https://2014.igem.org/Team:UCC\_Ireland
* 8. https://2020.igem.org/Team:HKUST
* 9. https://2024.igem.wiki/nau-china/
* 10. Wasserman, O.; Oliveira, P. E.; Bell, B. E.; Jefferson, S.; Fairbanks, S.; Watson, A.; Lewis, R. V.; Jones, J. A. Engineered Recombinant Hagfish Intermediate Filament Proteins: Unraveling Domain Roles in Synthetic Fiber Formation and Mechanics. ACS Omega 2024, 9 (47), 47023–47030.
* 11. Jones, R. J.; McFarland, S.; Rodgers-Ahnen, S.; Rhodes, R. Artemis Suit Material Project Overview, ICES-2025-56; NASA Johnson Space Center / KBR HHPC / Leidos: Houston, TX, 2025.
* 12. Sun, R. One-Step Assembly and Properties Study of Recombinant Hagfish Slime Proteins. Master’s Thesis, Beijing University of Chemical Technology, Beijing, China, 2023.

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{% block lead %}Detailed experimental protocols for plasmid extraction, PCR, electrophoresis, protein expression, purification, and characterization.{% endblock %}
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# Experiment

## Contents

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* [Plasmid Extraction](#header1)
* [PCR Amplification](#header2)
* [DNA Electrophoresis](#header3)
* [DNA Gel Recovery](#header4)
* [Gibson Assembly](#header5)
* [Bacterial Transformation](#header6)
* [Protein Expression](#header7)
* [Ultrasonication](#header8)
* [Protein Electrophoresis](#header9)
* [Protein Purification](#header10)

## Plasmid Extraction (TIANGEN Mini Plasmid Kit Protocol)

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### Preparations

* Ensure RNase A has been added to Buffer P1.
* Add the specified volume of absolute ethanol to Buffer PW.

### Steps

1. Column Equilibration: Add 500 µl Buffer BL to a CP3 Spin Column (in a Collection Tube). Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through. Place the column back in the tube.
2. Harvest Cells: Centrifuge 1-5 ml bacterial culture at 12,000 rpm for 1 minute. Discard the supernatant completely.
3. Resuspend Pellet: Add 250 µl Buffer P1 to the pellet. Resuspend completely by pipetting or vortex until no cell clumps remain.
4. Lysе Cells: Add 250 µl Buffer P2. Mix gently by inverting the tube 6-8 times. The solution should become clear and viscous. Do not vortex. Do not allow lysis to proceed for more than 5 minutes.
5. Neutralize: Add 350 µl Buffer P3. Mix immediately and gently by inverting 6-8 times. A white precipitate will form. Centrifuge at 12,000 rpm for 10 minutes.
6. Bind DNA: Transfer the supernatant from Step 5 to the equilibrated CP3 Spin Column. Avoid transferring any precipitate. Centrifuge at 12,000 rpm for 30-60 seconds. Discard the flow-through. Place the column back in the tube.
7. Wash (1): Add 600 µl Buffer PW to the column. Centrifuge at 12,000 rpm for 30-60 seconds. Discard the flow-through. Place the column back in the tube.
8. Wash (2): Repeat Step 7.
9. Dry Membrane: Centrifuge the empty column at 12,000 rpm for 2 minutes to dry it. Optional: Open the column lid and let it air-dry at room temperature for 2-5 minutes to ensure all ethanol evaporates (remaining ethanol may affect enzyme reaction, including PCR).
10. Elute DNA: Place the column in a clean 1.5 ml microcentrifuge tube. Add 50-100 µl Buffer EB (we use ddH2O, pH 7.0-8.5, as buffer to DNA sequencing) directly to the center of the membrane. Let it stand at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 2 minutes to collect the purified plasmid DNA.

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## PCR Amplification

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## DNA Electrophoresis

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## DNA Electrophoresis Recovery (TIANGEN Gel Purification Kit)

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## Gibson Assembly

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## Bacterial Transformation

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## Protein-induced Expression

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## Ultrasonication

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## Protein Electrophoresis

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## Protein Purification

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{% block title %}Results{% endblock %}
{% block lead %}Results of engineered fusion proteins combining hagfish intermediate filament (HIF) proteins with mussel foot proteins (MFPs) for space exploration applications.{% endblock %}
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# Results

## Contents

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* [Overview](#header1)
* [Results from Cycle 1: Design and Expression of Fusion Protein](#header2)
* [Results from Cycle 2: Expression Optimization of Fusion Proteins](#header3)
* [Results from Cycle 3: Functional and Performance Testing of Fusion Proteins](#header4)
* [Results from Cycle 4: Unstructured Domain Removal of rHIF Proteins](#header5)

## Overview

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To address the limitations of current materials for space exploration—specifically their lack of self-assembling, adhesive, and hydrogel-forming capabilities—a fusion protein was engineered by combining hagfish intermediate filament (HIF) proteins with mussel foot proteins (MFPs). Key outcomes are summarized below:

The fusion protein was successfully heterologously produced in *E. coli* BL21 (DE3) under the regulation of the conventional T7-LacO operon.

The resulting biomaterial forms threads and hydrogels with high water content, strong adhesiveness, and mechanical resilience.

Comprehensive assessments (protein yield, hydrogel formation, adhesive strength, and structural stability) confirmed its potential as a novel, multifunctional material for future space applications.

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## Results from Cycle 1: Design and Expression of Hagfish Intermediate Filament-Mussel Foot Fusion Protein

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## Results from Cycle 2: Expression Optimization of Fusion Proteins

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## Results from Cycle 3: Functional and Performance Testing of Fusion Proteins

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## Results from Cycle 4: Unstructured Domain Removal of rHIF Proteins

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