Synthetic biology approaches to exploring property-function relationships of reflective proteins for adaptive coloration and repellence

本研究的目的是利用现代合成生物学和重组DNA方法，建立(1)合成和表征新的反射素蛋白和(2)制备具有良好的光学、结构和适应性的反射素薄膜/纤维的方法。要实现这些目标，首先需要通过使用高通量组装、表达和结构/功能表征方法来快速探索序列-结构-功能空间，然后设计和利用基于反射素的薄膜/纤维和光子结构的制备和功能表征方法，来探索反射素的序列-性质-功能关系。首先，将开发反射素结构域的超变异和将反射素序列组装成全长基因的方法，这将包括折叠结构的预测、结构域边界的估计/探索以及保守氨基酸在确定光学性质和结构完整性中的作用的研究。然后，在使用自动组合组装方法形成全长结构并筛选所需的溶解性之前，将使用C-末端GFP标签来探测反射素变异域的溶解性。最后，将探讨非规范氨基酸(具有新的化学、物理和生物学特性的氨基酸)的正交引入，目的是将自然无法实现的新功能引入组装的反射素结构域，然后对这些合成的反射素蛋白进行功能分析。将探索和开发基于反射素的薄膜和纤维的制备方法，例如使用旋转法、浸渍和流动涂层以及溶胶凝胶法，目的是开发具有理想性能的材料，特别是自适应着色和排斥。

Using modern synthetic biology and recombinant DNA approaches, the aim of this study is to establish methods for (1) the synthesis and characterisation of novel reflectin proteins and (2) the fabrication of reflectin-based films/fibres with desirable optical, structural, and adaptive properties. Achieving these aims will require probing the sequence-property-function relationships of reflectins by firstly rapidly exploring the sequence-structure-functional space using high-throughput assembly, expression and structural/functional characterisation methods, and secondly, designing and utilising methods of fabrication and functional characterisation of reflectin-based films/fibres and photonic structures. Initially, methods for both the hyper-variation of reflectin domains and domain-domain assembly of reflectin sequences into full-length genes will be developed, which will involve predictions of folded structure, estimation/exploration of domain boundaries, and investigations into the role of conserved amino acids in defining optical properties and domain structural integrity. The solubility of reflectin variant domains will then be probed using C-terminal GFP tagging before full-length constructs are formed using automated combinatorial assembly methods and screened for desired solubility. Finally, both the orthogonal introduction of non-canonical amino acids (amino acids possessing novel chemical, physical, and biological properties) will be probed with the aim of introducing novel functionality, likely unattainable by nature, into assembled reflectin domains, followed by the functional analysis of these synthetic reflectin proteins. Methods of fabrication of reflectin-based films and fibres will be explored and developed, for example using spin-, dip, and flow-coating, and sol-gel processing, with the aim of developing materials with desirable properties, particularly adaptive colouration and repellence.