Engineering | NTU-Taiwan

Content

Introduction Design Phase

AI Sequence Generation Construct Design

Build Phase

Coloning Fermentation

Test Phase

Post-Processing Mechanical Properties

Learn Phase 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.

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:

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.
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.

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.

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

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

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.

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.

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

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.

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

IPTG induction is initiated when the bacterial growth reaches an OD600 of 50.
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.
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.

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.

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:

Dissolution: Recombinant spider silk protein from the T9-E9-TS9-S1 sequence is dissolved in HFIP.
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.
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.

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:

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.
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.
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.

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.

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.

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