Part:BBa_K1763444

Bombyx mori silk co-spinning module

Bombyx mori silk fibroin co-spinning module. Translational unit controlled by an IPTG inducible LacI promoter, Elowitz RBS, and a 6x polyhistidine tag for IMAC-purification of finalized construct. Contains the sfGFP protein domain for use as a reporting marker to verify functional co-spinning of silk domains.

While silk as a biomaterial is widely known for its remarkable mechanical strength, elasticity, and dexterity, the frontiers of silk engineering have been focused on refining and adding novel functions to silk peptides. Chemical modifications of the amino-acid sequences constructing silk fibroins have been performed to add a wide array of biochemical functions to regenerated silk structures. These functions vary from the ability to interact with nanoparticles for precision drug delivery, to seeding growth factors use as tissue scaffolds in medical applications, to even producing fluorescent silks for use in the fashion industry. Functionalizing silk has a potential to to develop highly sophisticated materials for novel synthetic biology applications.

However, functionalizing silk has yielded several difficulties that prevent widespread expansion and adoption of these novel technologies. Namely, the addition of large protein structures or functional domains in the repetitive monomer structure of the fibroin heavy and light chains has potential to disrupt the beta-sheets absolutely critical for proper fiber formation. Further, the ability to clone these repetitive structures with these large domains proves to be highly difficult, especially when it comes to gene assembly and oligo synthesis.

Previous work elucidating the structure of the N-terminal and C-terminal domains of Bombyx mori has revealed their critical nature in fiber assembly. The CTD of the chains are critical for producing disulfide-linkages to to other chains, while the NTD is responsible for protein trafficking and pH/mechanical stress mediate fiber formation through traditional spinning methods. This work suggests that the NTD-CTD structures are critical features of fiber assembly, and may allow for the delivery of internally inserted domains into the silk fiber.

Previous work has shown that a transgene encoding an eGFP flanked by the NTD and CTD structures transfected into Bombyx mori allow for the production of localized GFP in the silk glands of the silkworm, and are spun into the cocoons and silk threads themselves. These remarkable fluorescent silks reveal the ability to encode a functional domain in between the H-chain NTD/CTD, and have it express and function in thread form.

However, one of the difficulties in scaling this method for manufacturing and biomaterial use is the relative difficulty and length of time in rearing transgenic silkworms compared to tradition E. coli chassi for recombinant protein production. Production of recombinant E. coli proteins have been extensively analyzed and streamlined in the past century, with multiple laboratory strains and purification techniques developed to overexpress and generate high yields of desired proteins.

Here, we hypothesize that if the transgene produced previously can function in Bombyx mori silkworms, that the same recombinant protein can be produced and purified high yield from E. coli chassis, and used with natural silk dope in artificial spinning apparatuses. Our work has generated a Biobrick of this NTD-sfGFP-CTD construct, in which the functional domain can be easily swapped in the construct with proteins found in the Standard Registry of Parts with Silver Fusion prefixes and suffixes for in-frame protein protein fusions. We have shown that this BioBrick generates a protein of the proper size and yield, and that this protein, when spun artificially with Bombyx mori silk dope, produces a fiber with significant fluorescence, indicative of proper sfGFP function and immobilization with silk assembly structures.

Sequence and Features

Assembly Compatibility:

• 10
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• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1448
  Illegal BamHI site found at 221
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1439
  Illegal BsaI.rc site found at 719
  

Usage and Biology

The use of silk fibroin produced by both spiders and silkworms have recently arisen as a suitable alternative to synthetic fiber products for a wide variety of cosmetic, engineering, and biomedical opportunities. However, efforts to develop high-throughput production of silk fibroin via an in vivo approach have proven to be unsustainable and highly costly to process. Work to produce recombinant fibroin using high expression Escherichia coli chassis has shown to be both effective and scalable, but the difficulty in generating a large cloning library with monomeric MaSP1/2 subunits for polymerization with various affinity binding domains prevents the creation of modular silk fibroins that can perform an array of functions. To add modular and scalable affinity domains to nascent silk fibroins, a composite silk construct containing the N- and C- terminal domains derived from Bombyx mori silkworm silk constructions ligated to a reporting marker flanked by Type IIS restriction sites was cloned and expressed in E. coli colonies. Preliminary results indicate high expression levels driven by an IPTG-inducible promoter hours after induction, and IMAC purification indicates high degree of protein yield and purity via electrophoretic analysis. Further characterization must be conducted to verify protein conformation and function using blotting analysis, spectroscopy, and fluorescence assaying.

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

• https://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).

Background

Silk fibers produced by the arachnid Nephila clavipes (Golden Silk-Orb Weavers) and Bombyx mori produce unique mechanical and immunogenic properties (Vepari et al., 2007). The highly tensile and elastic properties of these fibers, coupled with the non- immunogenic and non-allergenic phenomena observed with spider silk, make it a suitable candidate for a wide variety of biomedical applications (Schacht et al., 2014). In particular, studies into the use of the spider silk monomers MaSP1 and MaSP2 with fusions to select molecules or interactive binding domains have arisen to study the potential for spider silk as a seeding and drug delivery mechanism (Humenik et al., 2011). However, large-scale production of spider silk has been hampered due to the poor sustainability of Golden Spider farming and subsequent silk collection procedures (Schiebel, 2004). Recent explorations into the use of recombinant silk proteins through bacterial chassis and microbiological vectors have shown tremendous promise, but are still hindered by a wide variety of issues, including (1) relatively low yield of recombinant protein relative to the amount of cell culture needed, (2) a complex and time-consuming process in constructing and troubleshooting modular forms of the silk-binding site fusions, (3) harsh chemical conditions during the purification of recombinant proteins that lead to potential denaturation of active peptides, (4) GC-rich hindrance of mRNA transcript stability and translational termination, and (5) poor scalability of recombinant silk production absent the use of large, relatively expressive bioreactors for bacterial fermentation (Winkler et al., 2000; Prince et al., 1995; Arcidiacono et al., 2002).

Studies investigating the potential for co-spinning, a process in which the terminal domains of the functional B. mori silk protein heavy chain are fused with genes encoding functional binding domains, and subsequently are combined with native silk proteins to form crystalline fibers, have shown some success (Sponner et al., 2005). In vivo co-spinning simulations have previously been conducted using the transgene in silkworms, producing functionally fluorescent N-terminal – GFP- C-terminal fusions that successfully incorporate into endogenous Bombyx mori silkworm silk monomers (Kojima et. al., 2007). These N-Terminal and C-terminal regions are not only easier to clone due to their non-repetitive structures, but additionally allows for storage and assembly of endogenous ampullate spidroins (Askarieh et al., 2010). Composite silkworm-spider silk chimeras have shown improved mechanical properties, and may increase the ease in high-yield production due to the easier manipulation of silkworm silk monomers (Teule, et. al. 2012). Yet, this process still relies on the use of transgenic silkworms, which have longer developmental cycles, require larger storage and nutrient conditions, and difficulty to harvest relative to bacterial counterparts (Murphy et al., 2009).

Construct Created

An ex situ co-spinning process has been developed for the production of composite B. mori silk fibers with functional protein fusions in bacterial chassis (namely, E. coli). This process results in the construction of a transgene that can be modularized using restriction enzymes to incorporate a wide range of functional peptide binding sites or other proteins, including avidin-biotin binding domains, RGD-cadherin motifs, albumin binding domains, or antibody affinity domains (Jansson et al., 2014). Specifically, this composite structure focuses on the ability to express functional sfGFP in silk fibers as a marker for successful co-spinning, and verifies proper co-spinning module expression using SDS-PAGE analysis.

A Standard Registry part encoding sfGFP (BBa_I746916) flanked by conserved B. mori N- and C- silk fibroin heavy chain terminal domains (NC-sfGFP) has been produced. This part is ligated in the standard registry pSB1C3 vector backbone, and transformed into chemically competent Escherichia coli cell cultures. Expression of the fusion protein is induced using IPTG, and extracts are purified using Immobilized Metal Affinity Chromatography (IMAC) using a Ni-NTA Sepharose resin bed designed to pull down polyhistidine tagged protein constructs.

Cloning Design of Construct

PCR Amplification

The terminal chimera construct was produced using traditional cloning. A gBlocks sequence containing the core structure of the DNA construct was ordered from IDT, and two primer sets designed to (1) amplify the core gBlocks sequences and (2) amplify and append the Standard Registry Biobrick prefix and suffix were ordered (Table 1).

The final gene construct contains, reading from 5’ to 3’: (1) Biobrick standard coding prefix, (2) an inverting regulator sensitive to CAP and LacI protein expression, creating an IPTG-inducible LacI promoter region (BBa_R0010), (3) ribosomal binding site (RBS - BBa_B0034), (4) a 6x polyhistidine tag flanking the (4) N-terminal region of the B. mori heavy chain fibroins and (5) C-terminal region of the B. mori heavy chain fibroins. A (6) reporter construct containing a superfolder GFP (BBa_I746916) was introduced in between the terminal domain constructs flanked by Type IIs restriction sites specific for BsaI cleavage activity (Table 2). This superfolder GFP was chosen primarily because of its in-vitro denature resistant properties and its fluorescent activity in visible light. Lastly (7), the construct is appended with a Biobrick suffix for standard assembly ligation into standard registry vectors. The standard registry vector pSB1A3 was selected for this construct, conferring an Ampicillin/Carbenicllin resistance gene and high copy nature (Table 3).

Constructs were ordered as an IDT gBlock and amplified using a Q5 Polymerase Reaction.

Component	Amount (for 50 uL reactions)	Final Concentration
5x Q5 Reaction Buffer	10 uL	1x
10 mM dNTPs	1 uL	200 uM
10 uM VF2	2.5 uL	0.5 uM
10 uM VR	2.5 uL	0.5 uM
Co-Spinning Module Template	0.5 uL of 1ng/uL stock	500 pg/50uL
Q5 DNA Polymerase	0.5 uL	0.02 U/uL
Nuclease Free ddH2O	33 uL	N/A

Templates were diluted from 10 ng/uL gBlocks stock by adding 1 uL of stock to 9 uL of MilliQ water in an eppendorf tube and stored at -20 degrees. 0.5 uL of the 1ng/uL stock was used for a final PCR concentration of 500 pg/50 uL (or 10pg/uL of reaction).

The annealing temperature calculated using the [http://tmcalculator.neb.com/#!/ NEB Q5 Tm calculator] was 66 deg Celsius, the same as the extension temperature. Below is the reaction conditions setup:

Step	Temp (degrees Celsius)	Time
Initial Denaturation	98	30 seconds
30 cycles	98	10 seconds
	66	20 seconds
	72	20 seconds (1kb amplicon)
Final Extension	72	2 minutes
Hold	4

After PCR was finished, the resulting products were mixed with 10 uL of 6x Loading Dye and split into two 30 uL products. Each sample was loaded into a 1% wt/vol agarose gel, along with 10 uL of 1 kbp GeneRuler DNA ladder, and ran for 20 minutes at 100V in 1x TAE buffer.

Digestion & Ligation Assembly into pSB1C3

Digestion

Component	50 uL Reaction
Template DNA (Co-Spinning Module, Plasmid pSB1C3)	1 ug
10x NEB 2.1 Buffer	5 uL (1x)
EcoRI	1 uL (10 Units)
PstI	1 uL (10 Units)
Nuclease free ddH2O	Up to 50 uL

Ligation

Component	20 uL Reaction
10x T4 DNA Ligase Buffer	2uL (1x)
Vector DNA (pSB1C3)	[http://nebiocalculator.neb.com/#!/ 1:3 molar ratio (pmol)]
Insert DNA (Co-Spinning Module)	1:3 molar ratio (pmol)
T4 DNA Ligase	1 uL
Nuclease Free ddH2O	up to 20 uL

Samples were loaded, mixed, and placed into the thermocycler. Samples were ligated for 10 minutes at RT (22 degrees Celsius), and heat inactivated at 65 degrees Celsius for 10 minutes, and held at 4 degrees Celsius indefinitely. Samples were then removed and chilled on ice, before being transformed into chemically competent E. coli BL21(DE3) Mix n Go cells.

Colony PCR Screening and Sequencing Verification of the BioBrick

Double Digestion Verification of Proper BioBrick Assembly

Protein Expression Strategy

Plasmid efficiency was qualitatively analyzed, and fluorescence of the colonies indicated that (1) the sequence was integrated into the genome of the colonies, and (2) that the construct was being prematurely expressed, indicating non-specific expression from the LacI promoter. Starter cultures required 6 hours of incubation to reach the 0.6 OD600 necessary for proper integration, in addition to the overnighting of the culture. Qualitative analysis indicates a gradient of fluorescence across all protein extracts and cell pellet samples between each hour, indicating enhanced gene expression following IPTG induction at each and every hour.

Protein Purification Strategy

Collection of several fractions of eluted protein through the IMAC column were pooled and analyzed using SDS-PAGE. Protein samples ran yielded a high degree of elution of a concentrated sample of protein using an initial elution in 250mM imidazole (Figure 3). With respect to the protein precision standards, the band of protein ranged between 50kDA and 70kDA, consistent with the estimated molecular weight of the NC-sfGFP construct at 58.59 kDA using GeneInfinity analysis. Initial cell lysate indicated presence of this protein in the initial flow through of the IMAC, indicating residual loss of protein sample through the column or oversaturation of the resin bed with histidine-tagged proteins. Successive wash steps using 40mM or 50mM imidazole buffers in a gradient fashion indicated little to no elution of protein, suggesting both that a higher concentration is needed to elute the tagged protein of interest, and that the initial flow through and primary water wash steps removed much of the trace lysate elements. Similarly, elution in 100mM imidazole yielded no trace protein samples visible using SDS-PAGE, suggesting higher imidazole concentrations are needed to outcompete the nickel chelating agents in the resin to release the NC-sfGFP construct. Initial elution of the protein sample of interest using 250mM imidazole qualitatively indicated a high release of protein sample through the gel, indicative of proper release and elution of fractionated protein sample using IMAC. Further elution using 250mM imidazole resulted in a lack of protein release in the SDS PAGE, suggesting total protein elution using the initial 250mM elution buffer. Protein precision dual color standards were used both as a kDA reference marker and a positive input control in the SDS-PAGE.

Quantification of Protein Yield using BCA Assaying

Methodology to co-spin the purified construct with B mori silk dope.

Stress-Strain and Fluorescence Analysis of Co-Spun Fiber

Future Directions and Potential Design Strategies with the Spun Fiber

Cloning of the composite silk structure suggests proper orientation and sequence generation of a novel BioBrick for submission in the Registry of Standard Parts. This BioBrick has been successfully transformed and expressed in DH5 cells, revealing non-specific induction of the gene from the LacI promoter under ultraviolet illumination. This suggests that while the NC-sfGFP construct is inducible under the LacI promoter, minimal protein expression is leaked in E. coli colonies.

Additionally, the Type IIS restriction sites in the NC-sfGFP construct have shown to be successful in inserting protein domains without the potential for frame-shift mutations using the Silver Fusion assembly 23 standard. This potential is suggested by the insertion of the Protein G Albumin Binding Domain, which was successfully inserted into the NC-composite gap using a simple digestion and ligation assembly method. This suggests the potential for the composite silk structure to be used as a chassis for delivery of multiple affinity domains in a scalable manner. This rapid assembly potential yields credence for the part to be extremely useful for multiple biomedical approaches in a synthetic biology context, where several binding domains of interests can be quickly ligated into the N and C terminal domains in a cost efficient and high-throughput manner.

Initial work in purification of the polyhistidine-tagged N-sfGFP-C protein indicates a high degree of specific elution at a 250mM imidazole concentration, indicating that fractionation of the sample in the 250mM elution buffer is sufficient for high recovery of silk proteins of interest. From a qualitative analysis, protein expression of the composite silk sequence appears to be enhanced after induction of IPTG, and every hour thereafter. This indication, however, has yet to be quantitatively analyzed via fluorescence analysis, colorimetric analysis of protein concentrations in each sample, and conformation and size verification of the protein construct.

With verification of the sfGFP marker expression and purification of the NC-sfGFP construct completed, there are several downstream proteomic experiments to be conducted to verify proper functioning of both the terminal domain regions, and the ability for the affinity domains to process ligand binding in a rapid and efficient manner. First, development of a NC-sfGFP hydrogel composite with B. mori solubilized silk would yield critical insight into the ability of the fused complex to form a stable gel matrix, with full expression of the affinity domain or marker of interest. Assaying for binding potential to the doped silk solution with the addition of stripping buffers may yield insight into the ability of agitation to harm hydrogel stability. Second, characterization of the composite protein for potential degradative activity in a wide variety of potential buffers used in materials fabrications can be conducted using Western blot analysis to assay for degradative products. Basic characterization of the protein can be analyzed using circular dichroism (CD), IR spectroscopy, and Cryo-EM for ultrastructural analysis of NC-sfGFP composite protein and varying gel meshes and pore size.

Intercalation of the N and C terminal domains with the albumin binding domain of Protein G may yield additional insight into the potential for the composite structure as a binding mechanism to common ligands found in biomaterials and tissues, including human serum albumin. Testing for enrichment of NC-ABD-silk hydrogels with a mixed solution including albumins may yield characterization insight into the ability for the affinity domains to selectively bind critical molecules, suggesting a role for the composite silk structures as a selectively permeable membrane for use in potential reconstruction matrices.

Additionally, binding strength of the N and C terminal regions may be analyzed with the silk monomers themselves to characterize binding selectivity to silk sequence regions, in addition to fluorescence following washes of the NC-sfGFP washes of the protein of interest to determine long term affinity domain function over time and in differing stressors.

Additional work must be done to verify stability of the composite protein over time. Long term storage of the proteins may be jeopardized by freeze thaw cycles and temperature-mediated degradation; addition of a glycerol medium or varying freezing temperatures may alleviate these issues. Lastly, data collection and analysis of the final composite silk construct must be conducted using Bradford and BCA assays, in conjunction with fluorescence analysis to quantity protein expression for initial BioBrick characterization.

Synthesis and purification of the NC-sfGFP suggests high potential for the ability to utilize composite silk structure systems for affinity binding and additional functionalization, resulting in the ability to utilize silk fusions in a wide variety of biomedical and cosmetic applications. These results yield promise for the application of these composite silks for use in future characterization.