Team:UCLA/Project/Functionalizing Silk

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BACKGROUND

METHODOLOGY

RESULTS

BIOBRICKS

Silk Functionalization: Developing the Next Generation of High Performance Fibers

Background

Abstract

Silk fibers possess the potential to be transformed into functional biomaterials that can be exploited in an array of biomedical applications, from aiding nanoscale drug delivery to simulating medical sutures. However, traditional methods of incorporating functional domains into fibers involve difficult, costly, and time-consuming processes. We propose an in vitro, co-spinning method to quickly and efficiently functionalize silk fibers. In essence, we spin a mixture of wild-type silk dope spiked with a small volume of functional domain. This functional domain which will bind to the native silk proteins when co-spun, thereby incorporating itself into the final synthetic fiber. To ensure proper binding of our functional domain, we created a co-spinning module. This module is a genetic construct consisting of our gene of interest flanked on either side by the N and C terminal domains of Bombyx mori (silkworm silk). When co-spun, the termini on our synthetic protein will bind to the respective termini in the native silk proteins, thereby functionalizing the fiber. Our goal is to develop, optimize and experimentally validate our co-spinning module, and assess its potential as a scalable and powerful tool to manufacture silk fibers with an array of functional capacities.

Bombyx mori larva
Wolinsky, National Geographic Creative

Bombyx mori silk proteins consist of sericin, a group of glycoproteins encapsulating fibroin. Fibroin serves as a vehicle for incorporating desired functionality.
Nato, Hiroshima University

Introduction

Native silk fibers exhibit great tensile strength, elasticity, and flexibility. These mechanical properties, coupled with the non-immunogenic behavior of silk proteins, render silk fibers a worthy candidate in the realm of biomedical applications. Due to the potential of these fibers as a vehicle for nanoscale drug delivery or a scaffold for tissue engineering, there is much interest in attaching domains onto these fibers to achieve desired functionality. Bombyx mori, commonly known as silkworm, contain silk proteins comprised of fibroin, the core fiber that provides silkworm silk it’s structure. Fibroin is the main protein of interest that we aim to attach functional domains onto. Fibroin serves as a vehicle for functionality.

Previously, to functionalize fibroin proteins, scientists have relied on chemical conjugation of silk peptides or breeding transfected silkworms that express transgenes encoding functional domains. However, these methods have inherent limitations. Attaching big, bulky functional domains directly onto the ends of fibroin proteins can disrupt Beta-sheet formation between the fibers. Preserving secondary structure is critical to preserve functionality. Moreover, native Bombyx mori silk genes are incredibly repetitive, which makes cloning of transgenes complex and time consuming. The breeding of transfected silkworms can be summed up as costly and laborious. These disadvantages prevent chemical conjugation and in vivo expression from becoming sustainable, cost-efficient ways of manufacturing silk fibers with functional capacities. As a result, there is much need to develop a new method to manufacture functional fibers.

Methodology

Fig 1: Our novel, co-spinning module, with our gene of interest, sfGFP, inserted between the non-repetitive N and C terminal domains of Bombyx mori, along with appropriate Bsa1 restriction sites. Also not shown is a 6X histidine tag, used for later protein purification processes.

The theory behind the co-spinning methodology is to attach a functional domain onto the native silk fiber without disrupting the natural mechanical properties or non-immunogenic behavior of wild-type silk. To achieve this, we've created a genetic construct, entitled our "co-spinning module", consisting of super folder Green Fluorescent Protein (sfGFP) flanked on either sides by the N and C terminal domains of Bombyx mori. These termini are critical in maintaining the structural integrity of the fibers. Specifically, the N terminal domains of individual fibroin proteins bind to their identical counterparts on adjacent fibroin proteins, thereby establishing disulfide linkages between the fibers. The C terminal domains on the fibroin proteins aid the fibers in responding to decreases in pH levels and mechanical stresses, conditions that induce the stacking of Beta sheets into an actual fiber. The repetitive motifs in between the terminal domains are hydrophobic regions that cluster together, separate from the hydrophilic N terminal domains, thus simulating a micelle. From our co-spinning module, we create a synthetic protein that emulates the composition of native fibroin, with the repetitive motifs between the domains replaced with our functional domain, sfGFP. When co-spun, the N terminal domains on our synthetic protein will recognize and bind to their counterparts on the native fibroin proteins, maintaining the micellar structure. The repetitive regions on the native fibroin and the sfGFP on our synthetic protein are coiled structures that, when exposed to the mechanical stresses induced by in vitro syringe extrusion, will flatten and stack up into Beta sheets, and Beta sheet formation is the key indication of proper fiber formation.

Fig 2: An outline of our expression techniques, using E-coli chassis. The 6X histidine tag attached to our co-spinning module enables us to use IMAC, immobilized metal affinity chromatography with nickel resin beads.

Fig 3: In our co-spinning methodology, Bombyx mori silk dope is spiked with a small volume (3 ul) of functional domain. The N termini on our expressed co-spinning module protein bind to the N-termini in the wildtype silk fibroin, and these hydrophilic heads arrange in a micellar structure, isolating the dangling coils of repetitive regions and sfGFP. After in-vitro syringe extrusion, the sheer forces applied induces Beta sheet formation.

Fig 4: Co-spinning module validated with our first, proof of concept co-spin of sfGFP with wild type Bombyx mori silk dope.

Results

Successful in vitro co-spin of wild type Bombyx mori silk dope with our expressed co-spinning module protein to produce a fiber that visibly fluoresces!

Fig 5: Our co-spun synthetic fiber under Evos FL digital fluorescence microscopy.

Achievements

1. Successful development and experimental validation of our co-spinning module as a tool to incorporate functional domains into wild-type Bombyx mori silk dope, with is then processed into a functional, synthetic fiber.
2. Designed and sequence-verified our novel co-spinning module with super folder GFP, (sfGFP) sandwiched between the N and C terminal domains of Bombyx mori.
3. Successful cloning of co-spinning module with E-coli chassis, and successful amplification of part with Polymerase Chain Reaction (PCR).
4. Successful expression and purification of sfGFP protein with N and C termini attached on either side.

Fig 6: DNA gel verifies successful cloning and PCR amplification of NC-sfGFP genetic construct. NC is an abbreviation for N and C terminal domains of Bombyx mori. A bright band at approximately 1650 kb, the expected insert size. Cloning and amplification done in both vectors, psB1A2 (first lane) and psB1C3 (third lane) yields comparable results.

Fig 7: SDS PAGE verifies successful expression and purification of our NC-sfGFP co-spinning module, with a singular, dark band at 66kD.

Future Directions

Now that we have established a proof of concept with sfGFP, you can imagine how we can swap sfGFP with other functional domains to spin out synthetic fibers that exhibit an array of functionality! In order to verify this BioBrick as a screening platform to assay for functional peptide affinity, a wide variety of co-spinning modules must be constructed. Namely, co-spinning modules digested and ligated with albumin-binding domain (ABD), immunoglobin G binding domain (ImGBD) and avidin binding domains (AvBD) may be of critical use in both experimental design and developing a functional application of co-spinning in a wide variety of biomedical applications.

Fig 8: Schematic outlining future functional domains to test, including albumin-binding domain (ABD), avidin binding domain (AvBD) and immunoglobin G binding (ImGBD).

List of Biobricks

• http://parts.igem.org/Part:BBa_K1763444

References

Teulé, F. et al. Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties. Proc. Natl. Acad. Sci. U.S.A. 109, 923–8 (2012).

Teulé, F. et al. A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning. Nat Protoc 4, 341–55 (2009).

Jansson, R. Strategies for Functionalization of Recombinant Spider Silk. 11, 76 (Acta Universitatis agriculturae Sueciae, 2015).

Kojima, K. et al. A new method for the modification of fibroin heavy chain protein in the transgenic silkworm. Biosci. Biotechnol. Biochem. 71, 2943–51 (2007).