Characterization of intrinsic properties of promoters
Brown, James Robert
ACS Synthetic Biology
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Rudge, T., Brown, J. R., Federici, F., Dalchau, N., Phillips, A., Ajioka, J., & Haseloff, J. (2015). Characterization of intrinsic properties of promoters. ACS Synthetic Biology https://doi.org/10.1021/acssynbio.5b00116
Accurate characterization of promoter behavior is essential for the rational design of functional synthetic transcription networks such as logic gates and oscillators. However, transcription rates observed from promoters can vary significantly depending on the growth rate of host cells and the experimental and genetic context of measurement. Further, in vivo measurement methods must accommodate variation in translation, protein folding and maturation rates of reporter proteins, as well as metabolic load. The external factors affecting transcription activity may be considered extrinsic, and the goal of characterization should be to obtain quantitative measures of the intrinsic characteristics of promoters. We have developed a promoter characterization method that is based on a mathematical model for cell growth and reporter gene expression and exploits multiple in vivo measurements to compensate for variation due to extrinsic factors. First, we used optical density and fluorescent reporter gene measurements to account for the effect of differing cell growth rates. Second, we compared the output of reporter genes to that of a control promoter using concurrent dual-channel fluorescence measurements. This allowed us to derive a quantitative promoter characteristic (ρ) that provides a robust measure of the intrinsic properties of a promoter, relative to the control. We imposed different extrinsic factors on growing cells, altering carbon source and adding bacteriostatic agents and demonstrated that the use of ρ values reduced the fraction of variance due to extrinsic factors from 78% to less than 4%. This is a simple and reliable method for quantitative description of promoter properties.
promoter, transcription, characterization, ratiometric, fluorescence, quantification, design, modeling
TJR was supported by a Microsoft Research studentship and EC FP7 Project No. 612146 (PLASWIRES) awarded to JH, JRB by a Microsoft Research studentship and internship, and FF by CONICYT-PAI/Concurso Nacional de Apoyo al Retorno de Investigadores/as desde el Extranjero Folio 8213002 7, and EPSRC grant EP/H019162/1 awarded to JH. JWA acknowledges the EPSRC and the Wellcome Trust for support.
EC FP7 CP (612146)
External DOI: https://doi.org/10.1021/acssynbio.5b00116
This record's URL: https://www.repository.cam.ac.uk/handle/1810/252902