Spider silk has the potential to be a useful biomaterial due to its high tensile strength and elasticity. It is also biocompatible and biodegradable, making it useful for wound dressings and sutures, tissue and bone scaffolds, vessels for drug delivery, and ligament and tendon replacements. In some studies where spider silk has been used to grow cells, the silk has promoted more cell growth than the control. However, it is difficult to obtain the high volume of silk needed for these undertakings on a large scale. Spiders are territorial and cannibalistic, so they cannot be easily farmed. Therefore, spider silk proteins are frequently produced in other organisms. E. coli is often used for spider silk production due to the relative ease of gene manipulation and the cost effectiveness of large-scale fermentation. However, due to the large protein size of the spider silk and the repeating amino acid motifs, there are some challenges with production in E. coli. Metabolic modeling is a way to model the metabolism of an organism and can help overcome some of the difficulties of spider silk production in E. coli by predicting metabolic engineering strategies. In this study, a metabolic modeling tool known as dynamic FBA predicted that ammonium is depleted during cell growth. Laboratory results confirmed that by adding additional ammonium to the medium, the E. coli cells experienced more cell growth and were able to produce more spider silk protein

Using silkworms as the potential host to spin spider silk-like fibers is an area of intense research world-wide. The conventional methods used to create transgenic silkworms hosting spider silk-like gene limits the incorporation of spider silk-like protein and do not improve the mechanical performance of the composite silkworm/spider silk fibers. In this dissertation, synthetic spider ampullate genes were incorporated into the precise site of the fibroin heavy chain or light chain using the latest genome editing technology CRISPR/cas9 guided non-homologous end joining as opposed to conventional random integration using transposon-based piggyBac system. These protocols, with extensive applicability to other silkworm researches, improved the content of spider silk-like protein in the transgenic silkworm/spider silk fibers, increases genetic stability in offspring, and improves the mechanical performance of the transgenic fibers compared to traditional methods. In addition, an enhanced green fluorescence protein (eGFP) was successfully incorporated into the fibroin light chain of silkworms using CRISPR/C as 9 initiated homologous recombination. The transgenic silkworm/spider fibers emitted strong green fluorescence under excitation. These results demonstrate that the we successfully developed a protocol to make silkworm as a host to spin spider silk-like fibers.

Spider silks are incredible natural materials that have a wide variety of properties that can rival or outperform even common synthetic materials like Nylon and Kevlar. As nature’s architects, orb-weaving spiders spin seven different silks that are used for very specific roles throughout the spider’s lifecycle. These silks are comprised of proteins called spidroins. Each of these spidroins has evolved to have properties such as strength and/or stretch that make these silks successful and highly adapted in their designated roles in web construction, prey capture and reproduction. This study involves the production of minor ampullate silk by genetically modifying the bacteria Escherichia coli. Minor ampullate is a lesser studied silk that is used for the first spiral of the orb web. This spiral is a template that the spider uses to finish the web and provides stability during the web construction. Minor ampullate silk is strong, however it does not stretch so it may be well-suited for certain applications such as ballistic materials. By producing and purifying different arrangements of minor ampullate silk protein, it is possible to learn how this protein can be expressed without using the spider itself. This investigation sheds light on how deviations in the protein sequence and motif arrangement can produce different properties, which can potentially be used to make new materials.

Although numerous spider fibroins have been reported, no known silk coating peptides have been discovered. We provide the first biochemical evidence for a spider coating peptide, called SCP-2, found on gumfooted lines, scaffolding joints and egg cases. The presence of this spider coating peptide on the fibers is supported by MS/MS analysis. Using quantitative real-time PCR analysis, we also demonstrate that SCP-2 has a flagelliform-restricted mRNA pattern of expression. Molecular modeling of the SCP-2 amino acid sequence predicts it adopts an alpha-helical structure that is amphipathic in nature. SCP-2, which can be extracted from fibers using water, is hypothesized to influence the mechanical properties of the silk fibers as well as serve a protective function for the threads. Based upon the restricted pattern of expression of SCP-2, our findings reveal novel insight regarding the glandular function of the flagelliform gland in . cob weaving spiders, suggesting it produces aqueous coating materials that are deposited on a wide range of different silk types. In addition, in an attempt to advance our understanding regarding silk gene transcription, our lab has developed the first antibody against the bHLH factor SGSF. SGSF has been implicated as a potential transcriptional regulator of silk gene transcription in spiders. Development of the anti-SGSF antibody was accomplished via the overexpression and purification of a fusion protein in bacteria, which consisted of the C-terminal region of SGSF fused to thioredoxin. Purified SGSF fusion proteins were injected into rabbits and the polyclonal antiserum was collected and tested by western blot analysis to determine the specificity of the immunological reagent. Western blot analyses revealed the anti-SGSF antiserum was capable of recognizing bacterially expressed SGSF in an efficient manner. Collectively, these studies lay the groundwork for future investigations involving the use of the antibody to determine the role of SGSF in silk transcription.

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Circadian rhythms are nearly ubiquitous and are responsible for timing biological processes and allowing for anticipation of regular changes in the environment. The internal clocks of most organisms have a period very close to 24 hours with little variation. Spiders, however, do not seem to follow this pattern. Both the fastest (18 hours) and slowest (29 hours) naturally-occurring clocks are found in spiders, and variation within a species can be orders of magnitude larger than that of previously studied animals. Circadian rhythms are assumed to be adaptive, yet little is known about their heritability in arthropods. Heritability is defined as the amount of phenotypic variation that can be attributed to genetic variation passed down from parent to offspring. Phenotype can be influenced by many complex factors including environmental effects, dominance of genetic sequences, and gene interactions. Because of these influences, the phenotypic characteristics of an individual can vary greatly, and it is often difficult to precisely identify what is truly heritable. Using spiders as a model system, we can exploit the extreme variation in circadian rhythms to investigate the potential contribution from heritability. Strong heritability would suggest that wide variation in circadian rhythms likely reflects high genetic variability in the species. Alternatively, the environment may have a greater contribution in this variation relative to the effects of heritability. To test this, we chose Parasteatoda tepidariorum, a common cobweb spider with a relatively short circadian period of 21.7 hours and intraspecific variation of more than 4 hours. To estimate the heritability of circadian rhythm, adult females were gathered with accompanying egg cases, and juveniles were raised from those cases. Six fundamental parameters of circadian rhythms were measured from the locomotor activity of adults and juveniles. Of those six, only one parameter differed between adults and juveniles: the onset of locomotor activity during the first five days when light cycles were present (Mann-Whitney U= 1814, p= 0.04). When all six circadian parameters were compared by regression of adults to respective offspring, none showed significant correlation. This indicates that variation in circadian rhythms was likely not caused by parental genetics, and that environmental factors, such as artificial light at night, may be the source of the extreme circadian rhythms seen in spiders. Another possible cause for this variation may be the presence of weak molecular circadian oscillators that are more sensitive to environmental factors than those in most other circadian systems.

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