Half-life measurements of chemical inducers for recombinant gene expression

Journal of Biological Engineering

Paolo Magni²  Maria Gabriella Cusella De Angelis²  Giuseppina Micoli¹  Michela Casanova²  Susanna Zucca²  Lorenzo Pasotti²  Nicolo’ Politi² 

[1] Centro di Ricerche Ambientali, IRCCS Fondazione Salvatore Maugeri, via Salvatore Maugeri 10, Pavia, Italy;Centro di Ingegneria Tissutale, Università degli Studi di Pavia, via Ferrata 3, Pavia, Italy

关键词: HSL;    ATc;    IPTG;    BioBrick™;    Whole-cell biosensors;    Synthetic biology;    Chemical inducers;    Degradation rate;   

Others  :  804813
DOI  :  10.1186/1754-1611-8-5

 received in 2013-07-25, accepted in 2014-01-11,  发布年份 2014

【 摘 要 】

Background

Inducible promoters are widely spread genetic tools for triggering, tuning and optimizing the expression of recombinant genes in engineered biological systems. Most of them are controlled by the addition of a specific exogenous chemical inducer that indirectly regulates the promoter transcription rate in a concentration-dependent fashion. In order to have a robust and predictable degree of control on promoter activity, the degradation rate of such chemicals should be considered in many applications like recombinant protein production.

Results

In this work, we use whole-cell biosensors to assess the half-life of three commonly used chemical inducers for recombinant Escherichia coli: Isopropyl β-D-1-thiogalactopyranoside (IPTG), anhydrotetracycline (ATc) and N-(3-oxohexanoyl)-L-homoserine lactone (HSL). A factorial study was conducted to investigate the conditions that significantly contribute to the decay rate of these inducers. Temperature has been found to be the major factor affecting ATc, while medium and pH have been found to highly affect HSL. Finally, no significant degradation was observed for IPTG among the tested conditions.

Conclusions

We have quantified the decay rate of IPTG, ATc and HSL in many conditions, some of which were not previously tested in the literature, and the main effects affecting their degradation were identified via a statistics-based framework. Whole-cell biosensors were successfully used to conduct this study, yielding reproducible measurements via simple multiwell-compatible assays. The knowledge of inducer degradation rate in several contexts has to be considered in the rational design of synthetic biological systems for improving the predictability of induction effects, especially for prolonged experiments.

【 授权许可】

   
2014 Politi et al.; licensee BioMed Central Ltd.

【 图 表 】

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