微生物活体操纵与可控装配新方法的构建及其应用

Microbe is extremely abundent in nature, the size of which has a very wide coverage from nano- to micro-scale making it suitable to be processed at multi-scale level as natural " building blocks " and "chassis cell". Based on the urgent need of micro/nano bio-manufacture of microorganism, four controlling methods- - i.e. molecular template, magnetic control, microfluidics, and bio- printing - - for the process suitable for microbe have been proposed to dip into the behavioral mode of microorganism and design new micro/nano functional materials by controlling directed movement and ordered arrangement of microorganism living cells. To our best knowledge, it is a promising and challenging project with originality in the field of microorganism..This project aims at developing new methods and techniques of micro/nano manufacture based on physical/chemical/biological principles as well as establishing new ways for controlled manipulation and controllable living microorganism "cell factory", especially focusing on exploring two new techniques-micro-fluidic and bio-printing. Through combinational and synergistic effort, it is expected be able to control the microorganism and its product from molecular to nano/micro level. Thus, the application prospect is extremely attractive, and it is highly promising to open up a new field of micro/nano manufacturing with living microorganism. .Specifically, using Saccharomyces cerevisiae and Escherichia coli as mode strains and Acetobacter xylins and Flavobacterium heparinum as function strains to investigate regulatory factors affecting the movement behavior of microorganism, nano-scale effect, surface/interfacial effect and biological effect during the biological manufacture process of microbial micro/nano robot self-assembly to reveal the underlying principle of microorganism orientation and mechanism of formation of fine structure at multi-level by inducing their unique biological function. .Hopefully, this new way will facilitate specific design of individual microenvironment, exploration of the growth, metabolism and behavior of microorganism. It is not only probable to study the behavior of the same microorganism in different micro environment, but also possible to reveal the interaction between different microbial individuals. Furthermore, optimization of culture medium for massive fermentation as well as assembly of the traditional orthogonal experiment analysis and response surface analysis into small chips can be achieved by designing and printing of the culture medium of microorganism. Finally, it will provide theoretical basis and technique support for the design of microorganism reactor and regulation of massive bio-refinery by multi-level construction of complex microorganism community.

自然界中微生物种类极为丰富，尺寸涵盖了纳米级与微米级，是可用于纳米、微米以及多层次跨尺度加工的天然"基本单元"和"底盘细胞"。本项目立足于构建用于定位操纵和可控装配微生物活体"细胞工厂"的新方法，重点开拓和发展微流控和(或)微生物打印技术该两项新技术。以酿酒酵母和大肠杆菌为模式菌种，木醋杆菌和肝素黄杆菌为功能菌种，诱发其特有的生物学功能，通过微生物微纳米机器人进行受控自组装，研究影响微生物的运动行为的调控因素，揭示微生物定位调控原理和多层次精细结构的形成机制。通过该方法可以设计特定的个性化微环境，探寻微生物的个体生长、代谢与行为模式。还可通过对微生物培养基的设计和打印，实现对大规模发酵过程的培养基优化，将传统的正交实验分析、响应面实验分析手段芯片化。此外，通过多层次组装，可设计和构筑复杂体系的微生物群落，为微生物反应器的设计、大规模生物炼制的调控提供理论依据和技术支撑。

以微生物为“基本单元”和“底盘细胞”，在纳米、微米以及多层次跨尺度上进行加工是微生物研究领域的重要前沿，在微生物反应器的设计、大规模生物炼制等方面具有重要意义。本项目基于微生物的生物制造的目的，以酿酒酵母和大肠杆菌为模式微生物，木醋杆菌和肝素黄杆菌为功能菌种，利用微生物的特异结构和多样功能进行仿生和调控，操纵微生物进行加工组装，建立了用于微生物定位操纵和可控装配的微流控技术和喷墨生物打印技术，初步实现了对微生物进行多层次不同尺度的调控。围绕项目研究目标，首先在图案化生物芯片的构建方面，通过光刻结合化学刻蚀的方法制备了不同尺寸的精细微米级有序图案化结构，以及双层嵌套式网格及条纹图案，用于调控微生物的有序分布及运动；其次在微生物可控模型的构建方面，通过外加磁场信号实现对磁性矿化的微生物运动的智能控制，以及通过程序化光信号实现对工程菌运动的程序化智能控制，分别建立了磁控和光控微生物运动的方法，并在纳米尺度上实现了次级代谢产物（如细菌纤维素）的可控有序排列；第三，构建了不同图案化的纤维素并将其应用于多种组织工程材料，包括肠组织工程材料、人工椎间盘生物材料及人工血管生物材料；最后在生物打印方面，基于高分子模板可控制备工艺，开发并搭建出基于气动原理的微滴喷射成形系统，并将气动喷头与电机助推式喷头组合，设计了组合式多喷头3D打印系统，经过不断优化并对打印环境进行无菌化控制，将其应用于高分子多孔支架与精细高分子基多孔膜板打印，以及多孔支架和微生物的混合打印。本项目围绕对微生物进行精确的定向运动诱导以及定位有序排列的核心问题进行了较深入的探索，初步实现了对微生物活体的操作与可调控的装配，并应用于构建组织工程材料，为进一步的应用开发奠定了基础。