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 of manufacturing functionalized fibers.

Iterative Capped Assembly (ICA) is a cloning method that is used to sequentially assemble long, repetitive DNA sequences. This technique was developed by Briggs et al. in 2012 as a method to assemble Transcription Activator-Like Effector Nucleases (TALENs) which are sequence specific DNA binding proteins that consist of multiple repetitive monomers. Each repeat monomer is responsible for binding to a specific nucleotide in the target sequence. Due to the repetitive nature of TALE genes, conventional PCR is unable to reliably amplify these sequences due to non-specific primer binding.

Although ICA was developed using TALE construction as a model problem, this technique can be used to construct long, repetitive DNA constructs in a directly controllable fashion. ICA assembles repetitive sequences one monomer unit at a time, while preventing the elongation of incomplete nucleotide chains. The full length sequence is flanked by unique primer annealing sites, which allows the PCR amplification of the final product. This entire process is performed using a solid substrate, which greatly facilitates the construction of long sequences.

This summer, we adapt the use of Iterative Capped Assembly (ICA) as a technique for cloning recombinant spider silks in a time-efficient and specific manner that is unparalleled by existing methods. Our experience shows that ICA can be used as a method for the quick assembly of spider silk genes.

Methodology

1. Iterative capped assembly is similar to Golden Gate assembly, which uses unique sticky ends to assemble gene fragments in a specific order. Whereas Golden Gate is a one-pot reaction with all the pieces ligated simultaneously, ICA is a more “controlled” variation where pieces are assembled one at a time. ICA relies on using 3 different versions of the monomer to be assembled, each of which has different sticky ends such that the monomers must be assembled in an A-B-C fashion. This prevents monomers from self-ligating. Type IIS restriction enzymes (such as BsaI), which cleave outside of the recognition site are used to generate these.



2. A basic biobrick for ICA consists of a gene monomer flanked by BsaI recognition and cleavage sites. While each of the core monomer is identical, the restriction sites are oriented such that digestion with BsaI yields distinct sticky ends for each of the three types of units. These pieces must be digested to reveal the sticky ends before assembly.
3. Accessory pieces required in ICA include the initiator, streptavidin coated beads, the terminator, and the capping oligos.
   1. The initiator is a dsDNA fragment made by annealing two ssDNA oligos together. The initiator is designed such that one end is biotinylated, for conjugation to streptavidin coated beads. The other end has a sticky overhang and is designed to anneal to the forward sticky end of the ‘A-type’ monomer unit. This end is 5’-phosphorylated to enable ligation. The initiator also contains a primer binding site that can be used for PCR amplification, as well as other accessory sequences such as affinity tags and the biobrick prefix.
   2. Streptavidin coated beads serve as a solid support for the elongating DNA chain during ICA. The biotinylated end of the initiator binds to streptavidin to anchor the nascent construct. The ability to physically separate the DNA from solution is needed due to repeated wash and ligation steps used during ICA.
   3. The terminator is constructed similarly to the initiator, but lacks biotinylation. One end of the terminator is compatible to the reverse sticky end of the ‘C-type’ monomer unit. This end is 5’-phosphorylated to enable ligation. The terminator also contains a primer binding site that can be used for PCR amplification, as well as the biobrick suffix.
   4. The capping oligos are comprised of a single 5’-phosphorylated ssDNA oligo that can form a stable stem loop structure with a unique sticky end. There are three distinct caps, each of which can bind to the A, B, or C sticky ends
4. In each extension step, the next sequential monomer (A, B, or C) is added onto the growing chain. Chains that failed to extend during the previous extension step are capped using a hair-pin oligo that prevents subsequent extension. These capped chains are still present in the mixture for the duration of ICA, but do not participate in any ligation event, and are not amplified in the final PCR. Each final construct is flanked by a biotinylated initiator oligo which allows immobilization onto streptavidin beads, and a terminator oligo. These two oligos provide primer annealing sites which can be used to amplify the sequence using conventional PCR.
5. A generalized workflow is demonstrated below:
   1. The initiator, terminator, and capping oligos are prepared ahead of time by mixing the relevant oligos and ramping down from 95 C to form the working oligos.
   2. Monomers of each type (A, B, C) are digested from plasmid with BsaI and purified prior to ICA. These are termed working monomers.
   3. The initiator is attached to the streptavidin coated beads.
   4. An ‘A-type’ monomer is ligated to the end of the initiator. Afterwards, any unreacted fragments, as well as the ligase are washed off.
   5. Next, the ‘B-type’ monomer is ligated to the end of the growing chain. In this reaction mixture, an ‘A-type’ cap is also included, to terminate any chains that failed to extend in the previous ‘A-type’ ligation. Again, unreacted fragments are removed by washing.
   6. Next, the ‘C-type’ monomer is ligated. This reaction mixture contains the ‘B-type’ cap.
   7. Next the ‘A-type’ monomer is ligated. This time, the ‘C-type’ cap is also included in the mixture.
   8. This proceeds in a repetitive fashion until the desired construct length is reached.
   9. The final constructed is eluted off the beads. The eluate is used as a template for PCR to amplify the construct. Only complete constructs that contain the initiator and terminator are amplified. Capped constructs do not amplify.
   10. The amplified construct can now be used for downstream cloning.

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
   1. Redesigned MaSp1 and MaSp2 monomers with modified sticky ends, and cloned these into biobricks.
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.
   1. Optimized ICA for use with spider silk genes to enable fast, efficient assembly of repetitive constructs.
4. Used ICA to assemble a variety of silk genetic constructs of different length and composition to examine their properties.
   1. Used ICA to create 10 different silk constructs ranging from 3-mers to 15-mers, and constructs with MaSp1 and MaSp2 in ratios of [1:2], [1:1] and [2:1].

List of Biobricks

• MaSp2 AB: BBa_K1763002
• MaSp2 BC: BBa_K1763003
• MaSp2 CA: BBa_K1763004
• MaSp2 SeqAB: BBa_K1763009
• MaSp1 AB: BBa_K1763010
• MaSp1 BC: BBa_K1763011
• MaSp1 CA: BBa_K1763012
• MaSp1 SeqAB2: BBa_K1763423
• M2-3(1C3): BBa_K1763424
• M2-3(T7): BBa_K1763425
• M2-6(1C3): BBa_K1763426
• M2-6(T7): BBa_K1763427
• M2-9(1C3): BBa_K1763428
• M2-9(T7): BBa_K1763429
• M2-12(1C3): BBa_K1763430
• M2-12(T7): BBa_K1763431
• M2-15(1C3): BBa_K1763432
• M2-15(T7): BBa_K1763433
• M1-9(1C3): BBa_K1763434
• M1-9(T7): BBa_K1763435
• M1-12(1C3): BBa_K1763436
• M1-12(T7): BBa_K1763437
• M1/2[2:1]-12(1C3): BBa_K1763438
• M1/2[2:1]-12(T7): BBa_K1763439
• M1/2[1:1]-12(1C3): BBa_K1763440
• M1/2[1:1]-12(T7): BBa_K1763441
• M1/2[1:2]-12(1C3): BBa_K1763442
• M1/2[1:2]-12(T7): BBa_K1763443

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.

FIGURES: I DON'T KNOW WHERE TO PUT THEM

Functionalizing Silk Fibers

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.

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.