Direct measurements of IPTG enable analysis of the induction behavior of E. coli in high cell density cultures

Microbial Cell Factories

Research

Josep López-Santín¹  Glòria Caminal¹  Alfred Fernández-Castané¹ 

[1] Departament d’Enginyeria Química, Unitat de Biocatàlisi Aplicada associada al IQAC (CSIC), Universitat Autònoma de Barcelona, Bellaterra, Spain;

关键词: IPTG transport;    Recombinant protein production;    Fed-batch;    Bistability;    Permease;   

DOI  :  10.1186/1475-2859-11-58

 received in 2012-03-02, accepted in 2012-04-03,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundThe E. coli lac operon and its components have been studied for decades, and lac-derived systems are widely used for recombinant protein production. However, lac operon dynamics and induction behavior remain the paradigm of gene regulation. Recently, an HPLC-MS-based method to quantify IPTG in the medium and inside the biomass has been established, and this tool may be useful to uncover the lack of knowledge and allow optimization of biotechnological processes.ResultsThe results obtained from the study of IPTG distribution profiles in fed-batch, high cell density cultures allowed discrimination between two different depletion patterns of an inducer from the medium to the biomass in E. coli-expressing rhamnulose-1-phosphate aldolase (RhuA). Moreover, we could demonstrate that active transport mediates the uptake of this gratuitous inducer. Additionally, we could study the induction behaviors of this expression system by taking into account the biomass concentration at the induction time.ConclusionsIn the bistable range, partial induction occurred, which led to intermediate levels of RhuA activity. There was a direct relationship between the initial inducer concentrations and the initial inducer transport rate together with the specific activity. A majority of the inducer remains in the medium to reach equilibrium with the intracellular level. The intracellular inducer accumulation was a further evidence of bistability of the lac operon.

【 授权许可】

Unknown   
© Fernández-Castané et al.; licensee BioMed Central Ltd. 2012. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.