Transgenic Silkworms Expressing Spider Silk via Targeted Integration
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Solution Overview
Problem
Current methods for producing transgenic silkworms that express synthetic spider silk proteins face challenges in achieving mechanical properties comparable to natural spider silk due to limitations in protein size, expression levels, and stability, as well as variations in composite silk fibers caused by random integrations and heterogeneity in transposon-based systems.
Innovation Solution
The use of an optimized CRISPR/Cas9 system for targeted integration of large synthetic spider silk genes into the silkworm genome, specifically into the introns of the fibroin heavy chain or light chain genes, under the control of endogenous promoters, ensuring stable expression and improved mechanical properties of the resulting composite silk fibers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If transposon-based systems are used for introducing synthetic spider silk genes, then gene expression is achieved, but random integrations cause heterogeneity and variability in composite silk fibers
Solution Approach 1:
The synthetic spider silk gene is divided into multiple copies (e.g., MaSp1-8 representing 8 repeats) to achieve sufficient expression while maintaining manageable integration. This segmentation allows the gene to be introduced as a modular unit that can be stably integrated without causing excessive heterogeneity.
Solution Approach 2:
The patent changes the integration mechanism from random transposon-based insertion to targeted CRISPR/Cas9-mediated integration at specific loci in the silkworm genome. This parameter change from random to targeted integration eliminates the heterogeneity problem while maintaining stable gene expression.
2Strength
If large synthetic spider silk genes are introduced to achieve native-sized protein production, then mechanical properties improve, but expression levels and stability become difficult to maintain
Solution Approach 1:
The synthetic spider silk gene is merged with the silkworm fibroin heavy chain gene by integrating it into an intron of the fibH gene. This merging ensures that the spider silk protein is expressed constitutively whenever the silkworm produces fibroin, thereby maintaining stable expression levels and reliability for large protein production.
Solution Approach 2:
The synthetic spider silk gene utilizes the silkworm's own fibroin production machinery and regulatory elements to drive its expression. The gene is placed under the control of the endogenous fibH promoter, allowing it to harness the silkworm's natural silk production capabilities to express the synthetic protein stably.
3Ease of manufacture
If conventional spinning methods using HFIP are used to create fibers from recombinant spider silk proteins, then fiber formation is achieved, but the process becomes toxic and expensive
Solution Approach 1:
The transgenic silkworms utilize their own natural spinning apparatus and water-based solvent system to extrude the composite silk fibers. The silkworm's spinneret and salivary gland machinery automatically process the synthetic spider silk protein along with silkworm fibroin, eliminating the need for external chemical coagulants like HFIP.
Solution Approach 2:
The patent changes the solvent system from organic (HFIP) to aqueous, leveraging the silkworm's natural water-based silk production mechanism. This parameter change from organic to aqueous solvent eliminates toxicity and reduces costs while maintaining fiber formation capability.
4Productivity
If small recombinant spider silk proteins (30-110 kDa) are produced, then expression is achieved in host systems, but the proteins are much smaller than native spider silk and lack sufficient mechanical properties
Solution Approach 1:
The patent introduces multiple copies (8 repeats) of the synthetic spider silk gene into the silkworm genome, resulting in excessive production of the protein relative to what would be produced by a single copy. This excessive action ensures that sufficient protein is accumulated to form fibers with native-like mechanical properties while maintaining efficient expression.
Data Source
AI summary
Transgenic silkworms stably expressing synthetic spider silk genes or composite silkworm/spider silk genes are disclosed. The exogenous spider silk genes are stably intergrated into a defined site of the fibroin heavy chain intron or a fibroin light chain intron of silkworms. Synthetic spider silk proteins and composite spider silk-silkworm genes and proteins are provided. The expression of exogenous spider silk genes is driven by the endogenous fibroin heavy chain promoter, improving the genetic stability of transgenic silkworms. The composite silkworm/spider silk fibers exhibit exceptional mechanical performance, compared to normal silkworm silk fibers and other transgenic silkworm fibers.