Direct measurements of IPTG enable analysis of the induction behavior of <it>E. coli</it> in high cell density cultures
2012
Fernández-Castané Alfred | Caminal Glòria | López-Santín Josep
<p>Abstract</p> <p>Background</p> <p>The <it>E. coli lac</it> operon and its components have been studied for decades, and <it>lac</it>-derived systems are widely used for recombinant protein production. However, <it>lac</it> 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.</p> <p>Results</p> <p>The 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 <it>E. coli</it>-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.</p> <p>Conclusions</p> <p>In 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 <it>lac</it> operon.</p>
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