Validation of a high-throughput fermentation system based on online monitoring of biomass and fluorescence in continuously shaken microtiter plates.
Validation of a high-throughput fermentation system based on online monitoring of biomass and fluorescence in continuously shaken microtiter plates.
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DOI：
10.1186/1475-2859-8-31
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发表时间：
2009-06-04
影响因子：
6.4
通讯作者：
Büchs J
中科院分区：
文献类型：
--
作者：
Kensy F;Zang E;Faulhammer C;Tan RK;Büchs J
最近报道的高通量发酵系统与在线测量，称为BioLector，其验证的先进版本。该技术结合了高通量筛选和高信息含量，通过在线监测连续振荡微量滴定板中的散射光和荧光强度。给出了光学测量、克隆和培养基筛选以及启动子表征的校准中的各种实例。细菌和酵母生物量浓度高达50 g/L细胞干重可以线性相关的散射光强度。在培养基筛选中，BioLector可以清楚地展示其检测不同生物量和产品产量的潜力，并推导出定量评估培养基和营养物质的比生长速率。由于不适当的缓冲液条件导致的生长抑制可以通过生长速率降低和NADH荧光暂时增加来检测。GFP作为报告蛋白可以很好地研究酵母菌在不同碳源下启动子的调控。对90个不同的表达GFP的多形汉逊酵母克隆的克隆筛选描绘了生长行为的广泛分布和GFP表达的甚至更强的分布。传质条件的重要性可以通过改变E.大肠杆菌培养物。不同的填充体积导致培养物生长和酸化的偏差，两者分别通过散射光强度和pH指示剂的荧光监测。BioLector技术是在工程反应条件下进行定量微发酵的非常有用的工具。利用该技术，可以从在线生物量和产物浓度直接推导出比产量和速率，这比现有技术（例如微板读取器或基于光电管的培养系统）优越上级。特别是，对高通量有强烈需求的应用，如克隆和培养基筛选和系统生物学，可以受益于其简单的处理，高定量信息含量和自动化能力。
An advanced version of a recently reported high-throughput fermentation system with online measurement, called BioLector, and its validation is presented. The technology combines high-throughput screening and high-information content by applying online monitoring of scattered light and fluorescence intensities in continuously shaken microtiter plates. Various examples in calibration of the optical measurements, clone and media screening and promoter characterization are given. Bacterial and yeast biomass concentrations of up to 50 g/L cell dry weight could be linearly correlated to scattered light intensities. In media screening, the BioLector could clearly demonstrate its potential for detecting different biomass and product yields and deducing specific growth rates for quantitatively evaluating media and nutrients. Growth inhibition due to inappropriate buffer conditions could be detected by reduced growth rates and a temporary increase in NADH fluorescence. GFP served very well as reporter protein for investigating the promoter regulation under different carbon sources in yeast strains. A clone screening of 90 different GFP-expressing Hansenula polymorpha clones depicted the broad distribution of growth behavior and an even stronger distribution in GFP expression. The importance of mass transfer conditions could be demonstrated by varying filling volumes of an E. coli culture in 96 well MTP. The different filling volumes cause a deviation in the culture growth and acidification both monitored via scattered light intensities and the fluorescence of a pH indicator, respectively. The BioLector technology is a very useful tool to perform quantitative microfermentations under engineered reaction conditions. With this technique, specific yields and rates can be directly deduced from online biomass and product concentrations, which is superior to existing technologies such as microplate readers or optode-based cultivation systems. In particular, applications with strong demand on high-throughput such as clone and media screening and systems biology can benefit from its simple handling, the high quantitative information content and its capacity of automation.
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影响因子：
5.1
作者：
Schulte A;Lorenzen I;Böttcher M;Plieth C
通讯作者：
Plieth C
影响因子：
3.8
作者：
Hermann, R;Walther, N;Büchs, J
通讯作者：
Büchs, J
影响因子：
3.8
作者：
Samorski, M;Müller-Newen, G;Büchs, J
通讯作者：
Büchs, J
影响因子：
3.2
作者：
Stöckmann, C;Losen, M;Büchs, J
通讯作者：
Büchs, J
影响因子：
2.7
作者：
Viaplana, E;Rebordosa, X;Villaverde, A
通讯作者：
Villaverde, A