生物活材料的研究主要集中在利用单一细菌生产生物膜、水塑料等体外应用。由于菌株尺寸较小,当其应用于体内时,容易发生逃逸,导致滞留效果较差。为解决这一难题,本研究借助大肠杆菌(Escherichia coli)表面展示系统(Neae),在两个菌株表面分别展示SpyTag和SpyCatcher,构建一种双菌"锁扣"型生物活材料生产系统。两菌株之间通过SpyTag和SpyCatcher的结合,发生原位交联,从而长时间滞留在肠道部位。体外实验表明两菌株混合几分钟后,会发生明显的沉降。此外,利用共聚焦成像和微流控平台进一步证明了该系统在流动状态下的粘附效果。最后,为了验证该系统在体内应用的可行性,小鼠连续3 d口服A菌(p15A-Neae-SpyTag/sfGFP)和B菌(p15A-Neae-SpyCatcher/mCherry),收集肠道组织进行冷冻切片染色。结果表明,相较于未结合菌株,该双菌系统能更多滞留在小鼠肠道,为生物活材料进一步的体内应用奠定了基础。
At present, the research of biological living materials mainly focuses on applications in vitro, such as using a single bacterial strain to produce biofilm and water plastics. However, due to the small volume of a single strain, it is easy to escape when used in vivo, resulting in poor retention. In order to solve this problem, this study used the surface display system (Neae) of Escherichia coli to display SpyTag and SpyCatcher on the surface of two strains, respectively, and constructed a double bacteria "lock-key" type biological living material production system. Through this force, the two strains are cross-linked in situ to form a grid-like aggregate, which can stay in the intestinal tract for a longer time. The in vitro experiment results showed that the two strains would deposit after mixing for several minutes. In addition, confocal imaging and microfluidic platform results further proved the adhesion effect of the dual bacteria system in the flow state. Finally, in order to verify the feasibility of the dual bacteria system in vivo, mice were orally administrated by bacteria A (p15A-Neae-SpyTag/sfGFP) and bacteria B (p15A-Neae-SpyCatcher/mCherry) for three consecutive days, and then intestinal tissues were collected for frozen section staining. The in vivo results showed that the two bacteria system could be more detained in the intestinal tract of mice compared with the non-combined strains, which laid a foundation for further application of biological living materials in vivo.