iGEM 2024 — Team ntu-taiwan

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### Content

[Introduction](#introduction)
[Design Phase](#design)

[AI Sequence Generation](#AI_Sequence_Generation)
[Construct Design](#Construct_Design)

[Build Phase](#build)

[Coloning](#Coloning)
[Fermentation](#Fermentation)

[Test Phase](#test)

[Post-Processing](#Post_Processing)
[Mechanical Properties](#Mechanical_Properties)

[Learn Phase](#learn)
[Reference](#reference)

Introduction

This page houses experimental results corresponding to the experimental overview page that showcases how each stage of SAC’s spider silk production workflow fits into the **Design** → **Build** → **Test** → **Learn cycle**. In the experimental overview page, we also provided links to specific sections of this page for better and more intuitive viewing.

![](https://static.igem.wiki/teams/5294/project/engineering/dbtl-cycle-cut.png)

Figure1. The engineering cycle of Design → Build → Test → Learn

Design: AI Sequence Generation & Construct Design

AI Sequence Generation

In this phase of our project, we focus on the design of the spider silk protein sequence using the
pET28a-T9E9TS9S1-mEGFP as our backbone. The design process begins with utilizing Generative Personalized Spider Silk (GPSS),
a software tool that generates a variety of DNA sequences tailored for our synthetic spider silk production. The selection
of these sequences is guided by two primary criteria:

1. **Structural Similarity to Natural Spider Silk**: The chosen DNA sequence must resemble natural spider silk proteins, which
   typically comprise both hydrophilic and hydrophobic amino acids. This structural diversity promotes the formation of micelles,
   which enhances the solubility of silk proteins[1]. Given the extensive number of sequences generated by GPSS, we carefully filter out
   those with significant structural deficiencies or excessive repetitive sequences that may hinder commercial synthesis.
2. **Feasibility of Gene Synthesis**: Recognizing that the remarkable properties of spider silk are largely due to its repetitive sequences[2],
   we must also consider the limitations of gene synthesis companies. Excessive repetition can complicate synthesis, so our design incorporates
   BioBrick restriction sites (SpeI and NheI) at both ends of the selected spider silk sequence. This allows for future modifications, enabling us
   to increase the number of repeat units, which is crucial for enhancing mechanical properties such as strength and elasticity.

To address the challenges posed by the highly repetitive nature of spider silk sequences—typically comprising around 90% repeat regions—we employed
the Primer Extension cloning method. This technique enables us to manually synthesize approximately 650 bp of DNA that cannot be produced using standard
synthesis methods. The hydrophilic nature of the amino acids at both the N-terminal and C-terminal regions facilitates their synthesis; however, the internal
repetitive sequences present more significant challenges.

| No. | T9-E9-TS9-S9 | T9-E9-TS9-S1 | T7-E7-TS7-S3 | T3-E3-TS3-S7 |
| --- | --- | --- | --- | --- |
| Hydrophobic | 28.94% | 39.89% | 31.98% | 36.18% |
| Molecular weight (kDa) | 165.2 kDa | 66.6 kDa | 79.1 kDa | 31.9 kDa |
| Attribute | Neutral | Acidic | Acidic | Acidic |
| DNA length (base pair) | 5577 bp | 2152 bp | 2589 bp | 912 bp |
| GC content | 76.26% | 62.85% | 60.02% | 48.90% |
| Alpha-helix content | 30.96% | 20.10% | 23.99% | 31.25% |
| Beta-Sheet Extended strand | 06.14% | 09.25% | 10.08% | 12.17% |
| Random coil | 62.90% | 70.74% | 65.93% | 56.58% |

**Table 1** Comparison of Different Physical Properties of Spider Silk DNA and Protein Structures Generated by Personalized Spider Silk (GPSS)

Construct Design

The secondary structure formed by these repetitive sequences is critical, as they favor beta-sheet formation, which contributes to the crystalline
packing and overall physical strength of the spider silk[3]. Therefore, we divided the sequence into two parts: a synthesizable N-terminal and C-terminal
region, followed by the extension of the repetitive sequence using Primer Extension. Ultimately, we combined these segments using Gibson Assembly,
successfully synthesizing a DNA sequence that incorporates the essential repetitive regions.

![](https://static.igem.wiki/teams/5294/project/engineering/combine.png)

**Figure 2** Scheme illustrating the mass synthesis of DNA repetitive sequences using the Primer Extension cloning method

For vector selection, we opted for **pET28a-T7-mEGFP** as our backbone. The high expression levels facilitated by the T7 promoter are advantageous
for spider silk production. The mEGFP serves as a reporter protein, allowing us to visually track and quantify the amount of spider silk produced.
Additionally, the plasmid features a His-tag, enabling purification through column chromatography should we encounter issues with protein solubility.
In instances where the silk proteins can form micelles similar to natural spider silk, we can utilize centrifugation for purification, further streamlining the process.

![](https://static.igem.wiki/teams/5294/project/engineering/construct.png)

**Figure 3** Expression cassettes of pET28a-T7-T9E9TS9S1-mEGFP

Build: Cloning, Validation & Fermentation

Coloning

Plasmid Backbone Construction

We constructed our plasmid backbone using **pET28a-T7-mEGFP**. The backbone was generated through double digestion with **NdeI** and **SalI**, successfully
yielding a backbone fragment of **5989 bp** and a non-target fragment of **4272 bp**. The 5989 bp backbone was isolated via gel extraction for further assembly

![](https://static.igem.wiki/teams/5294/project/engineering/image10.png)

**Figure 4** Isolation of the backbone pET28a-T7-mEGFP non-target fragment using double digestion

Target Sequence Assembly

Given the complexities of spider silk sequences, we divided our target into two segments:

* **N-Terminal Domain (NTD): 1261 bp / C-Terminal Domain (CTD): 954 bp**

![](https://static.igem.wiki/teams/5294/project/engineering/image9.png)
![](https://static.igem.wiki/teams/5294/project/engineering/image13.png)

**Figure 5** Extension of insert DNA N-Terminal Domain (NTD) and C-Terminal Domain (CTD) utilizing primer extension to elongate segments that cannot be artificially synthesized

Due to the repetitive nature of these sequences, commercial synthesis methods were not feasible. We utilized **Primer Extension**
and **PCR Extension** techniques to successfully amplify both fragments to their expected sizes. Following purification, we employed
**Gibson Assembly** to integrate these segments with the backbone vector.

Transformation and Verification

* We transformed the assembled plasmid into **NEB Stable *E. coli*** strains, which have knockout mutations in ***recA*** and ***endA*** genes, enhancing plasmid stability.
* **Double Digestion**: Confirmed the correct insertion of the target fragment using **XbaI** and **BsrGI**, identifying one colony with the expected digestion pattern.

![](https://static.igem.wiki/teams/5294/project/engineering/image16.png)

**Figure 6** Colony PCR confirmation of the successful insertion of two synthesized insert fragments into the pET28a-T7-mEGFP backbone

* **Colony PCR** was performed using **PrimeSTAR GXL DNA Polymerase** to amplify a **2160 bp** fragment, confirming the presence of the insert with a prominent band while accounting for minor non-specific bands due to the sequences' complexity.

Expression in BLR(DE3) Strain

The confirmed plasmid was then transformed into **BLR(DE3)** cells, chosen for their ***endA*** knockout which further stabilizes plasmid DNA during expression. This strain’s integration of **T7 RNA polymerase** enables efficient and controlled expression of the spider silk protein.

Small-Scale Induction

We initiated protein expression in flasks by adding **1 mM IPTG** once the **OD600** reached **0.6**. Samples were collected at intervals (0, 24, and 44 hours) for analysis.

* **SDS-PAGE** results showed a gradual increase in protein expression over time, with significant bands observed at 24 and 44 hours.
* A **Western Blot** analysis confirmed the presence of the spider silk protein, showing a major band at approximately **95 kDa**.

![](https://static.igem.wiki/teams/5294/project/engineering/image5.png)

**Figure 7** Qualitative analysis of protein expression post-induction using SDS-PAGE and Western blotting

* To further validate production, we employed fluorescence microscopy. The spider silk protein, fused with **GFP**, emitted green light upon excitation at **488 nm**, indicating
  successful intracellular expression. The results revealed micelle formation, confirming proper folding and assembly of the protein, critical for its functionality

![](https://static.igem.wiki/teams/5294/project/engineering/image8.png)

**Figure 8** Observation of the production status of T9E9TS9S1 spider silk protein using fluorescence microscopy (100X)

Fermentation & Purification

Large-Scale Fermentation

To scale up production, we utilized wastewater as a carbon source in a bioreactor setting.
Key parameters such as dissolved oxygen levels, pH, and temperature were meticulously monitored.

* The optical density (OD600) reached **50**, confirming adequate microbial density for effective protein induction.

![](https://static.igem.wiki/teams/5294/project/engineering/fer1.png)

**Figure 9** Fermentation conditions for BLR(DE3) knockout *endA* pET28a-T7-T9E9TS9S1-mEGFP

1. IPTG induction is initiated when the bacterial growth reaches an OD600 of 50.
2. An increase in pH indicates that the nutrients in the medium are nearing depletion, prompting the bacteria to utilize amino acids, which results in an alkaline shift. Consequently, we begin a fed-batch production strategy by introducing a feed solution.
3. After the feeding is completed, the nutrients in the bioreactor are exhausted, and the bacteria may start to utilize the proteins we have produced. Therefore, at this stage, the bioreactor fermentation process concludes.

Comparison of Wastewater and Commercial Carbon Sources in Fermentation

To achieve lower carbon emissions and sustainable development, we used wastewater as a carbon source in the fermentation process for mass-producing spider silk protein, comparing it to commercial carbon sources. While protein yield was slightly lower with wastewater,
its carbon footprint was also reduced. This fermentation strategy minimizes environmental pollution associated with spider silk protein production and adds value by repurposing wastewater for industrial use.

|  | Commercial carbon source | Wastewater |
| --- | --- | --- |
| Working Volume (L) | 3.5 | 3.5 |
| Protein Yield (g/L) | 3.5 | 3.1 |

**Table 2** Comparison of Spider Silk Protein Production Metrics Using Commercial Carbon Sources and Wastewater

Purification Process

Following fermentation, **sonication** was employed to lyse the cells, effectively releasing the spider silk proteins. Centrifugation at **12,000 xg** facilitated the separation of cell debris, allowing the micelle structures of the spider silk proteins to aggregate for purification.

![](https://static.igem.wiki/teams/5294/project/engineering/image4.png)

**Figure 10** Centrifugation of the T9E9TS9S1 spider silk protein with mEGFP fluorescence

* We optimized **SDS** concentrations during purification, ultimately selecting **0.5% SDS** to balance impurity removal and protein integrity
* The purified proteins were lyophilized for convenient storage

Protein Verification

Finally, we employed **SDS-PAGE** and **Western Blot** analyses to verify protein purity. The SDS-PAGE indicated a distinct band around 95 kDa, confirming the high purity of the spider silk protein and ensuring its suitability for downstream applications.

![](https://static.igem.wiki/teams/5294/project/engineering/image7.png)
![](https://static.igem.wiki/teams/5294/project/engineering/image3.png)

**Figure 11** Qualitative assessment of the purified T9E9TS9S1 spider silk protein using SDS-PAGE and Western blotting

Test: Post-Processing & Mechanical Properties

Post-Processing

Dissolving Purified Protein (HFIP)

Hexafluoro-2-propanol (HFIP) is employed as a solvent to dissolve our recombinant spider silk proteins from the T9-E9-TS9-S1 sequence.
Its unique ability to stabilize hydrophobic amino acids and the polypeptide backbone ensures that the proteins retain their structural integrity.
This results in a clear and stable solution, which is essential for preparing our materials for subsequent spinning and film fabrication[4].

Fiber Spinning Process

To produce spider silk fibers from the **T9-E9-TS9-S1** sequence, we follow a three-step process that mimics natural silk production:

1. **Dissolution**: Recombinant spider silk protein from the T9-E9-TS9-S1 sequence is dissolved in HFIP.
2. **Fiber Formation**: The protein solution is injected into an ammonium phosphate buffer (pH 4.3), which initiates an ion exchange process that promotes fiber formation. At this stage, the fibers are loosely arranged.
3. **Alignment and Strengthening:**: The fibers are treated in 95% isopropanol, aligning the protein molecules and enhancing their tensile properties through beta-sheet formation.

The resulting fibers are carefully examined to ensure uniform thickness and structure, critical for reliable mechanical testing.

![](https://static.igem.wiki/teams/5294/project/engineering/image2.jpg)

**Figure 12** Microscopic visualization of T9E9TS9S1 spider silk protein fibers

Film Fabrication Process

In addition to fibers, we also produce spider silk films using the following steps, also utilizing the **T9-E9-TS9-S1** sequence:

1. **Preparation of Spider Silk Solution**: A 5% (w/v) concentration of recombinant spider silk protein from the **T9-E9-TS9-S1** sequence is dissolved in HFIP. The powder is added slowly under gentle stirring to achieve a homogeneous solution.
2. **Casting**: The spider silk solution is cast into 2x3 cm Teflon molds. The non-stick properties of Teflon facilitate easy removal of the dried films.
3. **Drying**: The films are allowed to dry at room temperature for several hours, ensuring complete solvent evaporation and preventing residual solvent that could affect mechanical properties.

The dried films have a consistent thickness of 0.04 mm, which is crucial for reproducibility in mechanical testing.

![](https://static.igem.wiki/teams/5294/project/engineering/micro.png)

**Figure 13** Microscopic visualization of films produced from T9E9TS9S1 spider silk protein

Property Testing

Mechanical Testing

For mechanical property evaluation, we perform tensile testing on both fibers and films produced from the **T9-E9-TS9-S1** sequence:

* **Fiber Testing**: Specimens measuring 1 cm² with a width of 5 mm and a thickness of 0.04 mm are prepared. A universal tensile testing machine
  applies controlled force until the sample breaks, allowing us to measure ultimate tensile strength (UTS) and elongation at break. The fibers
  from the **T9-E9-TS9-S1** sequence show significantly higher tensile strength compared to films, indicating their suitability for applications
  requiring high durability.
* **Film Testing**: Similar specimens are cut from the films for tensile testing. Results demonstrate that while films exhibit lower tensile strength
  than fibers, they maintain strong correlations in strain and toughness with the fibers. This suggests that the fundamental mechanical properties of the
  protein are conserved across different material forms.

![](https://static.igem.wiki/teams/5294/project/engineering/strength.png)
![](https://static.igem.wiki/teams/5294/project/engineering/tough.png)
![](https://static.igem.wiki/teams/5294/project/engineering/strain.png)

**Figure 14** Tensile testing of silk and film produced from T9E9TS9S1 spider silk protein

X-ray Diffraction (XRD) Analysis of Spider Silk Proteins

To further investigate the structural properties of the spider silk proteins, we employed X-ray diffraction (XRD) analysis to assess the crystallinity of
the R1(Major ampullate spidroins 1, MaSP1), R2(Major ampullate spidroins 2,MaSP2), and T9E9TS9S1 spider silk protein. XRD is a powerful technique that provides
insights into the arrangement of atoms within a material, allowing us to determine the degree of crystallinity, which is directly related to a protein's physical
properties such as strength, elasticity, and durability.

In X-ray diffraction (XRD) analysis, the x-axis (2 theta angle) represents the diffraction angle (2θ), which corresponds to the angles at which X-rays are scattered
by the crystalline planes within the sample. The y-axis (intensity) reflects the strength of the X-ray signal detected after it is scattered, which is related to the
number of atomic planes contributing to the diffraction at a given angle.In this analysis, the diffraction patterns of the proteins were recorded and analyzed to determine
their crystalline and amorphous regions. The higher the crystallinity, the more ordered the molecular structure, which typically correlates with enhanced mechanical performance. Among the samples tested, the T9E9TS9S1 spider silk protein exhibited the highest degree of crystallinity, as indicated by sharper and more defined diffraction peaks. This suggests that T9E9TS9S1 spider silk protein has a more structured and tightly packed molecular arrangement compared to R1 and R2, which is likely responsible for its superior mechanical properties, such as increased tensile strength and toughness.

![](https://static.igem.wiki/teams/5294/project/engineering/peak.png)

**Figure 15** X-ray diffraction (XRD) analysis of spider silk proteins

Learn: Feedback Loop for AI Optimization

Our experiments have yielded significant conclusions regarding the production of spider silk fibers and films using the **T9-E9-TS9-S1** sequence.
The SAC system has proven to be fully functional, demonstrating the feasibility of transitioning from AI-generated sequences to actual spider silk
products. This successful proof of concept validates our entire process, from initial AI sequence generation to the final product.

However, we observed inconsistencies, particularly in property testing, where fluctuations in data were notably high. These variations can largely be attributed to human factors present in the purification and spinning phases, significantly impacting the reliability of our data and leading to variations in the mechanical properties of the final products.

A critical insight from our analysis involves the influence of the hydrophilic **mEGFP** reporter protein attached to the spider silk sequence. While mEGFP aids in tracking our protein during purification—allowing us to discern its presence in the pellet or supernatant—its hydrophilic properties may interfere with the beta-sheet structures that are crucial for the strength of the spider silk. This interaction could adversely affect the mechanical properties we aim to optimize.

On a positive note, our findings indicate that utilizing wastewater in conjunction with bioreactors effectively facilitates large-scale production. This underscores the progress of our SAC project toward industrial feasibility, highlighting the potential for sustainable and customized bioprocessing of unique materials like spider silk.

As we analyze the property testing data, we recognize considerable room for improvement in our experimental design. The SAC's **GPSS AI system** and the innovative use of **wastewater** have significantly contributed to our project, providing a solid foundation for developing a biomanufacturing approach that emphasizes customization, sustainability, and continuous learning.

Ultimately, the successful production of both spider silk fibers and films from the **T9-E9-TS9-S1** sequence underscores the exceptional strength and elasticity of spider silk. These qualities make it a promising biomaterial for diverse applications, particularly in tissue engineering and regenerative medicine. By refining our spinning and fabrication processes, we aim to harness the unique characteristics of spider silk, enhancing its potential for real-world applications.

Reference

* more information

  [1] Heim, M., Keerl, D., & Scheibel, T. (2009). Spider silk: from soluble protein to extraordinary fiber. Angewandte Chemie International Edition, 48(20), 3584-3596.

  [2] Adrianos, S. L., Teulé, F., Hinman, M. B., Jones, J. A., Weber, W. S., Yarger, J. L., & Lewis, R. V. (2013). Nephila clavipes flagelliform silk-like GGX motifs contribute to extensibility and spacer motifs contribute to strength in synthetic spider silk fibers. Biomacromolecules, 14(6), 1751-1760.

  [3] Van Beek, J. D., Hess, S., Vollrath, F., & Meier, B. (2002). The molecular structure of spider dragline silk: folding and orientation of the protein backbone. Proceedings of the National Academy of Sciences, 99(16), 10266-10271.

  [4] McLachlan, G. D., Slocik, J., Mantz, R., Kaplan, D., Cahill, S., Girvin, M., & Greenbaum, S. (2009). High‐resolution NMR characterization of a spider‐silk mimetic composed of 15 tandem repeats and a CRGD motif. Protein Science, 18(1), 206-216.

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