Creating Functional 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.

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

1. 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 folded 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 that 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.

Results

Using ICA, we have generated 10 silk constructs. These include constructs of pure MaSp2 ranging from 3-15 mers, pure MaSp1 of 9 and 12-mers and 12-mers of MaSp1/2 hybrids in 3 different ratios.

Future Directions

While we were able to construct many sequences of varying length and composition using ICA, we were unable to explore its maximum potential for cloning repetitive genes. We have not established an upper limit on the number of monomers able to be assemble using ICA. In addition, we have not explored the extended use of ICA to create extremely large (greater than 50 monomer units)

Achievements

1. Successfully improved last year’s biobrick BBa_K1384000
2. Created a collection of parts to be used for Iterative Capped Assembly
3. Demonstrated that ICA is adaptable to silk using our designed sticky ends.
4. Used ICA to assemble a variety of silk genetic constructs of different length and composition to examine their properties.

List of Biobricks

• MaSp2 AB: BBa_K1763002

References

Briggs A., Rios X., Chari R., Luhan Y., Zhang F., Mali P., and Church G. Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. Nucleic Acids Research. 2012;40(15): e117

Hinman, M.B., Lewis, R. Isolation of a clone encoding a second dragline silk fibroin. Nephila clavipes dragline silk is a two-protein fiber. J. Biol. Chem. 1992;267: 19320–19324.

Tokareva O., Michalczechen-Lacerda V., Rech E., and Kaplan D. Recombinant DNA production of spider silk proteins. Microbial Biotechnology. 2013;6(6): 651-663

Xu, M., Lewis, R.V. Structure of a protein superfiber: spider dragline silk. PNAS;1990;87, 7120.

Achievements

Successful in vitro co-spin of wild type Bombyx mori silk dope with synthetic protein to produce a fiber that visibly fluoresces! Designed and sequence-verified our novel co-spinning module with super folded GFP, (sfGFP) sandwiched between the N and C terminal domains of Bombyx mori. Successful cloning of co-spinning module with E-coli chassis, and successful amplification of part with Polymerase Chain Reaction (PCR). Successful expression and purification of sfGFP protein with N and C termini attached on either side.