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

蜘蛛丝是一种很有前途的天然材料，在工程和生物医学中有广泛的应用，从智能纺织品到组织工程的生物相容性支架。然而，天然丝蛋白有技术局限性，例如难以从其本地生产者那里获得足够数量的丝蛋白，或者它们的序列太大而不能进行异源表达(即在不同的生物体中生产)。此外，最常被研究的蜘蛛丝-牵引丝-在机械强度方面进行了优化，这不一定是生物材料应用中最理想的特性。此外，在不损害天然丝绸的许多吸引人的特性的情况下，对天然丝绸进行改性以引入额外的功能是具有挑战性的。为了克服这些限制，人们设计了迷你蜘蛛：这些重组丝绸的尺寸比天然丝绸小得多，但仍保留了许多令人满意的机械性能。然而，以前设计和表达微型蜘蛛的方法在范围上受到限制，研究往往只从狭窄的丝素序列池中提取，并调查具有相对较窄生物物理特性范围的少量微型蜘蛛。为了解决这个问题，我们需要(A)从本地蜘蛛基因组中新发现更多可能的丝蛋白，(B)更好地了解丝素序列-功能关系，(C)更快的系统来表征和快速迭代优化重组丝素，以及(D)更好的纺丝方法，这将是为蜘蛛丝定制的。在这个项目中，我们将通过以下方式实现这些目标：(A)应用机器学习策略来利用蜘蛛研究中的RNAseq数据来识别新的丝素序列模块；(B)利用合成生物学和实验的统计设计来系统地筛选大量嵌合微型蜘蛛的机械和可纺性特性，并建立蜘蛛功能设计的预测规则；(C)通过大量测试可纺性指标和具有深度流变学特征的纤维自组装机制来进行高通量、有针对性的可纺性分析；(D)应用工程的设计-建造-测试-学习循环，建立一条迭代的高通量管道，以开发能够纺成具有良好纺纱性能的丝纤维的蜘蛛。该项目的成果将使研究人员能够快速生产具有可预测性能的新的定制蜘蛛丝变体，大大扩大这种多功能材料在技术和经济上可行的应用范围。这将有助于通过与我们来自工艺创新和Spinex工程有限公司的行业顾问的密切互动来支持对蜘蛛丝的新兴生物技术开发，他们将提供实物支持。因此，应对人造丝工程的挑战需要范式的改变，这不只是从天然材料本身获得灵感，也不是将生物丝作为唯一的黄金标准，而是通过一种综合的仿生战略来推进，承认序列和加工需要协同进化的事实。我们假设，这种集成的方法将开辟通往新型人造丝绸的途径，这些人造丝绸自组装成目前无法到达的仿生层级结构，并赋予纤维性能改进。为此，我们将结合强大的序列设计和预测建模方法、更强大的丝蛋白表达系统，以及以前所未有的吞吐量组装、纺纱和生物物理表征丝库的创新技术，从而形成一个人造丝绸制造的综合平台：SPINS，一条研究新型蜘蛛的合成生物学管道。

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.