Smith, Charlotte Rose
(2022)
Spider silk: from super strong webs to customisable synthetic protein.
PhD thesis, University of Nottingham.
Abstract
Spider silks display a remarkable array of properties, making them an area of great interest in biotechnological research. Silk has many of the desired properties needed for a biomaterial; it is flexible, biocompatible and can have remarkable tensile strength, which makes it as strong and tough as some man-made materials such as Kevlar and steel. This combination of physical properties and biocompatibility makes it an especially interesting candidate for both bioengineering and medical applications. Due to this, and the relative difficulty of farming spiders due to their cannibalistic nature, much interest has focussed on the recombinant production of synthetic spider silk.
Unusual silks also have the potential to be invaluable in the pursuit of unique and tailored biomaterials. For example, the water spider Argyroneta aquatica uses its silk to create a diving bell, from where it spends most of its life submerged. The silk used to create this structure is gas permeable, which makes it an interesting case for further investigation. By learning from nature, we can hope to recapitulate these complex materials, adapting and applying them to biotechnological, medical, or industrial applications.
In this thesis, three bodies of work have been undertaken to further the knowledge of natural spider silk and the production of silk mimics in the laboratory. Each research chapter explores the diverse theme of spider silk in extreme environments. This research has created a pipeline for the identification, characterisation and translation of unusual natural silks into recombinant proteins.
Using both short and long read RNA sequencing technology, Chapters 2 and 3 investigate the amino acid makeup of the silk of spider species which live in extreme abiotic environments. Chapter 2 presents the first sequenced silk genes from linyphiid and nesticid spiders and the first genetic evidence that these spiders use at least four of the most well-characterised silk types, as well as uncovers a potentially novel silk type. It is shown here, using transcriptomic assemblies, that the silks used by spiders in acidic environments assemble in the expected way, while harbouring some diverged repetitive region motifs which may be key to their function.
Chapter 3 uses SMRT long read sequencing to present the first full length silk gene sequences from the water spider, Argyroneta aquatica, and the colonial tent-weaver, Cyrtophora citricola. In addition, short read RNA sequencing is used to create several robust transcriptomes from a range of spiders and tissue types. These sequences reveal environment and species specific silk adaptations, and proposes that specific acidic and basic residues could be important environmental adaptations. This long read data set also highlights the presence of frameshifts in silk sequences, which could be a possible novel way of naturally increasing spidroin diversity at the genetic level.
Recent technological advancements have enabled the recombinant production of artificial miniaturised silk proteins in Escherichia coli, and Chapter 4 uses the knowledge gained from natural silk transcripts to design and express a specialised A. aquatica spidroin. Several techniques are used, including Analytical Ultra Centrifugation and Mass Spectrometry, to characterise the dimerisation properties of the C- terminal domain, highlighting the importance of the repetitive region in fibre formation. This research demonstrates the utility of a modular approach to expressing recombinant silk proteins, and several novel constructs are designed to represent other silk types.
These studies highlight several approaches for the investigation into the silk genes of spiders that live in usual environments, and shows that the principle of recombinant production of miniaturised spidroins can be applied to more diverse silk types, to create different proteinaceous materials. With further refining, this pipeline could be used to efficiently design and create a range of functionalised recombinant spidroins, using natural spidroins as direct inspiration, with properties tailored for use as silkbased biomaterials.
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