Charrier Marimikel, Li Dong, Mann Victor R, Yun Lisa, Jani Sneha, Rad Behzad, Cohen Bruce E, Ashby Paul D, Ryan Kathleen R, Ajo-Franklin Caroline M
ACS Synth Biol. 2019 Jan 18;8(1):181-190. doi: 10.1021/acssynbio.8b00448. Epub 2019 Jan 7.
Materials synthesized by organisms, such as bones and wood, combine the ability to self-repair with remarkable mechanical properties. This multifunctionality arises from the presence of living cells within the material and hierarchical assembly of different components across nanometer to micron scales. While creating engineered analogues of these natural materials is of growing interest, our ability to hierarchically order materials using living cells largely relies on engineered 1D protein filaments. Here, we lay the foundation for bottom-up assembly of engineered living material composites in 2D along the cell body using a synthetic biology approach. We engineer the paracrystalline surface-layer (S-layer) of Caulobacter crescentus to display SpyTag peptides that form irreversible isopeptide bonds to SpyCatcher-modified proteins, nanocrystals, and biopolymers on the extracellular surface. Using flow cytometry and confocal microscopy, we show that attachment of these materials to the cell surface is uniform, specific, and covalent, and its density can be controlled on the basis of the insertion location within the S-layer protein, RsaA. Moreover, we leverage the irreversible nature of this attachment to demonstrate via SDS-PAGE that the engineered S-layer can display a high density of materials, reaching 1 attachment site per 288 nm. Finally, we show that ligation of quantum dots to the cell surface does not impair cell viability, and this composite material remains intact over a period of 2 weeks. Taken together, this work provides a platform for self-organization of soft and hard nanomaterials on a cell surface with precise control over 2D density, composition, and stability of the resulting composite, and is a key step toward building hierarchically ordered engineered living materials with emergent properties.
生物合成的材料，如骨骼和木材，兼具自我修复能力和卓越的机械性能。这种多功能性源于材料中活细胞的存在以及不同组分在纳米到微米尺度上的分级组装。虽然制造这些天然材料的工程类似物越来越受到关注，但我们利用活细胞对材料进行分级排列的能力很大程度上依赖于工程化的一维蛋白质细丝。在这里，我们使用合成生物学方法为沿细胞体在二维空间中自下而上组装工程化的生物活性材料复合材料奠定了基础。我们对新月柄杆菌的类晶体表面层（S层）进行工程改造，使其展示SpyTag肽，该肽能在细胞外表面与经SpyCatcher修饰的蛋白质、纳米晶体和生物聚合物形成不可逆的异肽键。通过流式细胞术和共聚焦显微镜，我们表明这些材料与细胞表面的附着是均匀、特异且共价的，并且其密度可以根据S层蛋白RsaA内的插入位置进行控制。此外，我们利用这种附着的不可逆性，通过SDS-PAGE证明工程化的S层可以展示高密度的材料，每288纳米达到1个附着位点。最后，我们表明量子点与细胞表面的连接不会损害细胞活力，并且这种复合材料在2周内保持完整。综上所述，这项工作为软、硬纳米材料在细胞表面的自组装提供了一个平台，能够精确控制所得复合材料的二维密度、组成和稳定性，是构建具有新兴特性的分级有序工程化生物活性材料的关键一步。
