Synthetic biology Pipeline for the Investigation of Novel Spidroins (SPINS)

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Spider silk is a promising natural material for a diverse range of applications in engineering and biomedicine, ranging from smart textiles to biocompatible scaffolds for tissue engineering. However, natural silk proteins have technological limitations; such as being difficult to harvest in sufficient quantities from their native producer or their sequences being too large for heterologous expression (i.e. production in a different organism). Moreover, the most commonly investigated spider silk - dragline silk - is optimised for mechanical strength, which is not necessarily the most desirable property for biomaterial applications. In addition, it is challenging to modify natural silks to introduce additional functionality without compromising on many of the attractive properties of native silk. In an attempt to overcome these limitations, mini-spidroins have been designed: these are recombinant silks much smaller in size than native silk that nevertheless retain many of its desirable mechanical properties. However, previous approaches to the design and expression of mini-spidroins have been limited in scope, with studies often only drawing from a narrow pool of silk sequences and investigating small numbers of mini-spidroins with a relatively narrow range of biophysical properties. To address this, we need (a) a larger range of possible silks proteins newly identified from the native spider genome, (b) a better understanding of silk sequence-function relationships, (c) faster systems for the characterisation and rapid iterative optimization of recombinant silks, and (d) a better spinning method which will be bespoke for spider silk. In this project we will achieve these aims by (a) applying machine-learning strategies to exploit RNAseq data from spider studies for the identification of new silk sequence modules; (b) using synthetic biology and statistical design of experiments to systematically screen large libraries of chimeric mini-spidroins for their mechanical and spinnability properties and establishing predictive rules for spidroin functional design; (c) conducting high-throughput, targeted spinnability analysis through bulk testing of spinnability indicators and fibre self-assembly mechanisms with in-depth rheological profiling; (d) applying the Design-Build-Test-Learn cycle of engineering to establish an iterative high-throughput pipeline to develop spidroins capable of being spun into silk fibres that possess favourable spinning properties. The results of this project will allow researchers to rapidly produce new custom-made spider silk variants with predictable properties, considerably expanding the scope of technically and economically viable applications of this versatile material. This will serve to support the emerging biotechnological exploitation of spider silk, through the close interaction with our industrial advisors from the Centre for Process Innovation and Spintex Engineering Ltd, who will give in-kind support.Thus, addressing the challenges of artificial silk engineering requires a change in paradigm, which is not just drawing inspiration from the natural material itself, nor using the biological silk as the sole gold standard, but proceeds through an integrated biomimetic strategy that acknowledges the fact that sequence and processing need to evolve in concert. We hypothesise that such an integrated approach will open up routes towards novel artificial silks that self-assemble into biomimetic hierarchical structures that are currently inaccessible and endow improved fibre properties. To this end we will combine powerful approaches to sequence design and predictive modelling, more robust silk protein expression systems, and innovative technologies for the assembly, spinning and biophysical characterisation of silk libraries at unprecedented throughput, resulting in a comprehensive platform for artificial silk manufacture: SPINS, a Synthetic biology Pipeline for the Investigation of Novel Spidroins.