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

利用现代合成生物学和重组DNA方法，本研究的目的是建立以下方法：(1)合成和表征新型反射蛋白；(2)制造具有理想光学、结构和自适应特性的反射蛋白基薄膜/纤维。实现这些目标将需要通过首先使用高通量组装、表达和结构/功能表征方法快速探索序列-结构-功能空间来探测反射体的序列-属性-功能关系，其次，设计和利用基于反射的薄膜/纤维和光子结构的制造和功能表征方法。首先，将开发反射蛋白结构域的高度变异和反射蛋白序列的结构域-结构域组装成全长基因的方法，这将涉及折叠结构的预测，结构域边界的估计/探索，以及研究保守氨基酸在定义光学性质和结构域完整性方面的作用。然后，在使用自动组合组装方法形成全长构建体并筛选所需的溶解度之前，将使用c端GFP标记来探测反射蛋白变异域的溶解度。最后，将探索非规范氨基酸（具有新的化学、物理和生物特性的氨基酸）的正交引入，目的是将自然界可能无法实现的新功能引入组装反射蛋白结构域，然后对这些合成反射蛋白进行功能分析。将探索和开发反射基薄膜和纤维的制造方法，例如使用自旋、浸渍和流动涂层以及溶胶-凝胶处理，目的是开发具有理想性能的材料，特别是适应性着色和驱避性。

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.