AVT, Biochemical Engineering, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.
Microb Cell Fact. 2009 Aug 1;8:42. doi: 10.1186/1475-2859-8-42.
In industry and academic research, there is an increasing demand for flexible automated microfermentation platforms with advanced sensing technology. However, up to now, conventional platforms cannot generate continuous data in high-throughput cultivations, in particular for monitoring biomass and fluorescent proteins. Furthermore, microfermentation platforms are needed that can easily combine cost-effective, disposable microbioreactors with downstream processing and analytical assays.
To meet this demand, a novel automated microfermentation platform consisting of a BioLector and a liquid-handling robot (Robo-Lector) was sucessfully built and tested. The BioLector provides a cultivation system that is able to permanently monitor microbial growth and the fluorescence of reporter proteins under defined conditions in microtiter plates. Three examplary methods were programed on the Robo-Lector platform to study in detail high-throughput cultivation processes and especially recombinant protein expression. The host/vector system E. coli BL21(DE3) pRhotHi-2-EcFbFP, expressing the fluorescence protein EcFbFP, was hereby investigated. With the method 'induction profiling' it was possible to conduct 96 different induction experiments (varying inducer concentrations from 0 to 1.5 mM IPTG at 8 different induction times) simultaneously in an automated way. The method 'biomass-specific induction' allowed to automatically induce cultures with different growth kinetics in a microtiter plate at the same biomass concentration, which resulted in a relative standard deviation of the EcFbFP production of only +/- 7%. The third method 'biomass-specific replication' enabled to generate equal initial biomass concentrations in main cultures from precultures with different growth kinetics. This was realized by automatically transferring an appropiate inoculum volume from the different preculture microtiter wells to respective wells of the main culture plate, where subsequently similar growth kinetics could be obtained.
The Robo-Lector generates extensive kinetic data in high-throughput cultivations, particularly for biomass and fluorescence protein formation. Based on the non-invasive on-line-monitoring signals, actions of the liquid-handling robot can easily be triggered. This interaction between the robot and the BioLector (Robo-Lector) combines high-content data generation with systematic high-throughput experimentation in an automated fashion, offering new possibilities to study biological production systems. The presented platform uses a standard liquid-handling workstation with widespread automation possibilities. Thus, high-throughput cultivations can now be combined with small-scale downstream processing techniques and analytical assays. Ultimately, this novel versatile platform can accelerate and intensify research and development in the field of systems biology as well as modelling and bioprocess optimization.
在工业和学术研究中，对具有先进传感技术的灵活自动化微发酵平台的需求日益增加。然而，到目前为止，传统平台无法在高通量培养中连续生成数据，特别是在监测生物量和荧光蛋白方面。此外，需要能够将具有成本效益的一次性微生物反应器与下游处理和分析测定轻松结合的微发酵平台。
为了满足这一需求，成功构建并测试了一种由 BioLector 和液体处理机器人（Robo-Lector）组成的新型自动化微发酵平台。BioLector 提供了一种培养系统，能够在微孔板中根据定义的条件永久监测微生物生长和报告蛋白的荧光。在 Robo-Lector 平台上编程了三种示例性方法，以详细研究高通量培养过程，特别是重组蛋白表达。在此研究了表达荧光蛋白 EcFbFP 的宿主/载体系统 E. coli BL21(DE3) pRhotHi-2-EcFbFP。借助“诱导曲线”方法，可以同时以自动化方式进行 96 种不同的诱导实验（诱导剂浓度从 0 到 1.5 mM IPTG 变化，在 8 个不同的诱导时间）。“生物量特异性诱导”方法允许在微培养板中以相同的生物量浓度自动诱导具有不同生长动力学的培养物，这导致 EcFbFP 产量的相对标准偏差仅为 +/- 7%。第三种方法“生物量特异性复制”可在具有不同生长动力学的预培养物中生成具有相同初始生物量浓度的主培养物。通过从不同的预培养微孔板的适当接种体积自动转移到主培养板的相应孔中来实现这一点，随后可以获得相似的生长动力学。
Robo-Lector 在高通量培养中生成广泛的动力学数据，特别是在生物量和荧光蛋白形成方面。基于非侵入式在线监测信号，可以轻松触发液体处理机器人的动作。机器人和 BioLector（Robo-Lector）之间的这种相互作用将高含量数据生成与系统的高通量实验以自动化的方式结合在一起，为研究生物生产系统提供了新的可能性。该平台使用具有广泛自动化可能性的标准液体处理工作站。因此，现在可以将高通量培养与小规模下游处理技术和分析测定相结合。最终，这种新型多功能平台可以加速和深化系统生物学领域的研究与开发以及建模和生物过程优化。
