The Ties that Stick: Challenges and Future Promise in the Field of Bioadhesives.

Bioadhesives are wet or dry polymeric compounds that rely upon physical and chemical properties to generate characteristic sticky forces. The past decade has seen a rapidly evolving field of research around the functions, genetics, biochemistry, and mimetics of bioadhesives, but challenges unique to this research area continue to arise. We polled the presenters of SICB Symposium #8 to describe the "Ties that Stick:" challenges and exciting prospects that most resonated with their research pursuits. Themes that emerged from the poll included difficulties working with adhesive-producing organisms, field inherent knowledge gaps in theoretical modeling, molecular interactions, technology, and the interdisciplinary rigor of the bioadhesives field. We address each challenge with a discussion of the opportunities and applications presented by bioadhesives research.

Repeat Variation Resolves a Complete Aggregate Silk Sequence of Bolas Spider Mastophora phrynosoma.

Many species of spider use a modified silk adhesive, called aggregate glue, to aid in prey capture. Aggregate spidroins (spider fibroins) are modified members of the spider silk family; however, they are not spun into fibers as are their solid silk relatives. The genes that encode for aggregate spidroins are the largest of the known spidroin genes and are similarly highly repetitive. In this study, we used long read sequencing to discover the aggregate spidroin genes of the toad-like bolas spider, Mastophora phrynosoma, which employs the glue in a unique way, using only a single, large droplet to capture moths. While Aggregate Spidroin 1 (AgSp1) remains incomplete, AgSp2 is more than an extraordinary 62 kb of coding sequence, 20 kb longer than the longest spidroin on record. The structure of repeats from both aggregate silk proteins follows a similar pattern seen in other species, with the same strict conservation of amino acid residue number for much of the repeats' lengths. Interestingly, AgSp2 lacks the elevated number and groupings of glutamine residues seen in the other reported AgSp2 of a classic orb weaving species. The role of gene length in glue functionality remains a mystery, and thus discovering length differences across species will allow understanding and harnessing of this attribute for the next generation of bio-inspired adhesives.

The next generation of biopanning: next gen sequencing improves analysis of bacterial display libraries.

BACKGROUND: Bacterial surface display libraries are a popular tool for novel ligand discovery due to their ease of manipulation and rapid growth rates. These libraries typically express a scaffold protein embedded within the outer membrane with a short, surface-exposed peptide that is either terminal or is incorporated into an outer loop, and can therefore interact with and bind to substrates of interest. RESULTS: In this study, we employed a novel bacterial peptide display library which incorporates short 15-mer peptides on the surface of E. coli, co-expressed with the inducible red fluorescent protein DsRed in the cytosol, to investigate population diversity over two rounds of biopanning. The naive library was used in panning trials to select for binding affinity against 3D printing plastic coupons made from polylactic acid (PLA). Resulting libraries were then deep-sequenced using next generation sequencing (NGS) to investigate selection and diversity. CONCLUSIONS: We demonstrated enrichment for PLA binding versus a sapphire control surface, analyzed population composition, and compared sorting rounds using a binding assay and fluorescence microscopy. The capability to produce and describe display libraries through NGS across rounds of selection allows a deeper understanding of population dynamics that can be better directed towards peptide discovery.

Toward Spider Glue: Long Read Scaffolding for Extreme Length and Repetitious Silk Family Genes AgSp1 and AgSp2 with Insights into Functional Adaptation.

An individual orb weaving spider can spin up to seven different types of silk, each with unique functions and material properties. The capture spiral silk of classic two-dimensional aerial orb webs is coated with an amorphous glue that functions to retain prey that get caught in a web. This unique modified silk is partially comprised of spidroins (spider fibroins) encoded by two members of the silk gene family. The glue differs from solid silk fibers as it is a viscoelastic, amorphic, wet material that is responsive to environmental conditions. Most spidroins are encoded by extremely large, highly repetitive genes that cannot be sequenced using short read technology alone, as the repetitive regions are longer than read length. We sequenced for the first time the complete genomic Aggregate Spidroin 1 (AgSp1) and Aggregate Spidroin 2 (AgSp2) glue genes of orb weaving spider Argiope trifasciata using error-prone long reads to scaffold for high accuracy short reads. The massive coding sequences are 42,270 bp (AgSp1) and 20,526 bp (AgSp2) in length, the largest silk genes currently described. The majority of the predicted amino acid sequence of AgSp1 consists of two similar but distinct motifs that are repeated ∼40 times each, while AgSp2 contains ∼48 repetitions of an AgSp1-similar motif, interspersed by regions high in glutamine. Comparisons of AgSp repetitive motifs from orb web and cobweb spiders show regions of strict conservation followed by striking diversification. Glues from these two spider families have evolved contrasting material properties in adhesion (stickiness), extensibility (stretchiness), and elasticity (the ability of the material to resume its native shape), which we link to mechanisms established for related silk genes in the same family. Full-length aggregate spidroin sequences from diverse species with differing material characteristics will provide insights for designing tunable bio-inspired adhesives for a variety of unique purposes.

Properties of orb weaving spider glycoprotein glue change during Argiope trifasciata web construction.

An orb web's prey capture thread relies on its glue droplets to retain insects until a spider can subdue them. Each droplet's viscoelastic glycoprotein adhesive core extends to dissipate the forces of prey struggle as it transfers force to stiffer, support line flagelliform fibers. In large orb webs, switchback capture thread turns are placed at the bottom of the web before a continuous capture spiral progresses from the web's periphery to its interior. To determine if the properties of capture thread droplets change during web spinning, we characterized droplet and glycoprotein volumes and material properties from the bottom, top, middle, and inner regions of webs. Both droplet and glycoprotein volume decreased during web construction, but there was a progressive increase in the glycoprotein's Young's modulus and toughness. Increases in the percentage of droplet aqueous material indicated that these increases in material properties are not due to reduced glycoprotein viscosity resulting from lower droplet hygroscopicity. Instead, they may result from changes in aqueous layer compounds that condition the glycoprotein. A 6-fold difference in glycoprotein toughness and a 70-fold difference in Young's modulus across a web documents the phenotypic plasticity of this natural adhesive and its potential to inspire new materials.

The impact of UVB radiation on the glycoprotein glue of orb-weaving spider capture thread.

Many spider orb-webs are exposed to sunlight and the potentially damaging effects of ultraviolet B (UVB) radiation. We examined the effect of UVB on the viscoelastic glycoprotein core of glue droplets deposited on the prey capture threads of these webs, hypothesizing that webs built by species that occupy sunny habitats are less susceptible to UVB damage than are webs built by species that prefer shaded forest habitats or by nocturnal species. Threads were tested shortly after being collected in the early morning and after being exposed to UVB energy equivalent to a day of summer sun and three times this amount. Droplets kept in a dark chamber allowed us to evaluate post-production changes. Droplet volume was unaffected by treatments, indicating that UVB did not damage the hygroscopic compounds in the aqueous layer that covers droplets. UVB exposure did not affect energies of droplet extension for species from exposed and partially to mostly shaded habitats (Argiope aurantia, Leucauge venusta and Verrucosa arenata). However, UVB exposure reduced the energy of droplet extension in Micrathena gracilis from shaded forests and Neoscona crucifera, which forages at night. Only in L. venusta did the energy of droplet extension increase after the dark treatment, suggesting endogenous molecular alignment. This study adds UVB irradiation to the list of factors (humidity, temperature and strain rate) known to affect the performance of spider glycoprotein glue, factors that must be more fully understood if adhesives that mimic spider glycoprotein glue are to be produced.

Temperature mediates the effect of humidity on the viscoelasticity of glycoprotein glue within the droplets of an orb-weaving spider's prey capture threads.

Sticky viscous prey capture threads retain insects that strike araneoid orb-webs. The threads' two axial fibers support a series of glue droplets, each featuring a core of adhesive viscoelastic glycoprotein covered by an aqueous solution. After sticking, the glue extends, summing the adhesion of multiple droplets, and dissipates some of the energy of a struggling prey. As a day progresses, threads experience a drop in humidity and an increase in temperature, environmental variables that have the potential to alter thread and web function. We hypothesize that thread droplets respond to these opposing environmental changes in a manner that stabilizes their performance, and test this by examining threads spun by Argiope aurantia, a species that occupies exposed, weedy habitats. We confirmed that decreased humidity increases glycoprotein viscosity and found that increased temperature had the opposite effect. To evaluate the combined effect of temperature and humidity on a droplet's ability to transfer adhesive force and dissipate energy, we extended a droplet and measured both the deflection of the axial line supporting the droplet and the duration of its tensive load. The cumulative product of these two indices, which reflects the energy required to extend a droplet, was greatest under afternoon (hot and dry) conditions, less under morning (cool and humid) conditions, and least under hot and humid afternoon conditions. Although the opposing effects of temperature and humidity tend to stabilize glycoprotein performance, A. aurantia thread droplets appear to function optimally during the afternoon, equipping this species to capture large orthopterans, which are most active at this time.