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This article is part of the supplement: Italian Society of Bioinformatics (BITS): Annual Meeting 2011

Open Access Research

Characterization of an inducible promoter in different DNA copy number conditions

Susanna Zucca12, Lorenzo Pasotti12, Giuliano Mazzini3, Maria Gabriella Cusella De Angelis2 and Paolo Magni12*

Author affiliations

1 Dipartimento di Informatica e Sistemistica, Università degli Studi di Pavia, Via Ferrata 1, I-27100 Pavia, Italy

2 Centro di Ingegneria Tissutale, Università degli Studi di Pavia, Via Ferrata 1, I-27100 Pavia, Italy

3 Istituto di Genetica Molecolare - Consiglio Nazionale delle Ricerche, Via Ferrata 9, I-27100 Pavia, Italy

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Citation and License

BMC Bioinformatics 2012, 13(Suppl 4):S11  doi:10.1186/1471-2105-13-S4-S11

Published: 28 March 2012

Abstract

Background

The bottom-up programming of living organisms to implement novel user-defined biological capabilities is one of the main goals of synthetic biology. Currently, a predominant problem connected with the construction of even simple synthetic biological systems is the unpredictability of the genetic circuitry when assembled and incorporated in living cells. Copy number, transcriptional/translational demand and toxicity of the DNA-encoded functions are some of the major factors which may lead to cell overburdening and thus to nonlinear effects on system output. It is important to disclose the linearity working boundaries of engineered biological systems when dealing with such phenomena.

Results

The output of an N-3-oxohexanoyl-L-homoserine lactone (HSL)-inducible RFP-expressing device was studied in Escherichia coli in different copy number contexts, ranging from 1 copy per cell (integrated in the genome) to hundreds (via multicopy plasmids). The system is composed by a luxR constitutive expression cassette and a RFP gene regulated by the luxI promoter, which is activated by the HSL-LuxR complex. System output, in terms of promoter activity as a function of HSL concentration, was assessed relative to the one of a reference promoter in identical conditions by using the Relative Promoter Units (RPU) approach. Nonlinear effects were observed in the maximum activity, which is identical in single and low copy conditions, while it decreases for higher copy number conditions. In order to properly compare the luxI promoter strength among all the conditions, a mathematical modeling approach was used to relate the promoter activity to the estimated HSL-LuxR complex concentration, which is the actual activator of transcription. During model fitting, a correlation between the copy number and the dissociation constant of HSL-LuxR complex and luxI promoter was observed.

Conclusions

Even in a simple inducible system, nonlinear effects are observed and non-trivial data processing is necessary to fully characterize its operation. The in-depth analysis of model systems like this can contribute to the advances in the synthetic biology field, since increasing the knowledge about linearity and working boundaries of biological phenomena could lead to a more rational design of artificial systems, also through mathematical models, which, for example, have been used here to study hard-to-predict interactions.