Section: New Results

Analysis of fluorescent reporter gene data

The use of fluorescent and luminescent reporter genes allows real-time monitoring of gene expression, both at the level of individual cells and cell populations (Section 3.2). Over the years, many useful resources have appeared, such as libraries of reporter strains for model organisms and computer tools for designing reporter plasmids. Moreover, the widespread adoption of thermostated microplate readers in experimental laboratories has made it possible to automate and multiplex reporter gene assays on the population level. This has resulted in large time-series data sets, typically comprising ${10}^5-{10}^6$ measurements of absorbance, fluorescence, and luminescence for ${10}^3$ wells on the microplate. In order to fully exploit these data sets, we need sound mathematical methods to infer biologically relevant quantities from the primary data and computer tools to apply the methods in an efficient and user-friendly manner.

In the past few years we developed novel methods for the analysis of reporter gene data obtained in microplate experiments, based on the use of regularized linear inversion. This allows a range of estimation problems to be solved, notably the inference of growth rate, promoter activity, and protein concentration profiles. The linear inversion methods, published in Bioinformatics in 2015 [12], have been implemented in the Python package WellFARE and integrated in the web application WellInverter . Funded by a grant from the Institut Français de Bioinformatique (IFB), we improved WellInverter by developing a parallel computational architecture with a load balancer to distribute the analysis queries over several back-end servers, a new graphical user interface, and a plug-in system for defining high-level routines for parsing data files produced by microplate readers from different manufacturers. This has resulted in a scalable and user-friendly web service providing a guaranteed quality of service, in terms of availability and response time. This year the web service has been redeployed on the new IFB cloud and on an Inria server, accompanied by extensive user documentation, online help, and a tutorial. Moreover, we submitted a journal paper on WellInverter illustrating the use of the tool by analyzing data of the expression of a fluorescent reporter gene controlled by a phage promoter in growing Escherichia coli populations. We notably show that the expression pattern in different growth media, supporting different growth rates, corresponds to the pattern expected for a constitutive gene.

Compared to most reporter gene assays based on fluorescence proteins, luciferase reporters have a superior signal-to-noise ratio, since they do not suffer from the high autofluorescence background of the bacterial cell. At the same time, however, luciferase reporters have the drawback of constant light emission, which leads to undesired cross-talk between neighbouring wells on a microplate. To overcome this limitation, Marco Mauri in collaboration with colleagues from the Philipps-Universität Marburg developed a computational method to correct for luminescence bleed-through and to estimate the “true” luminescence activity for each well of a microplate. As the sole input our algorithm uses the signals measured from a calibration plate, in which the light emitted from a single luminescent well serves as an estimate for the “light-spread function”. We show that this light-spread function can be used to deconvolve any other measurement obtained under the same technical conditions. Our analysis demonstrates that the correction preserves low-level signals close to the background and shows that it is universally applicable to different kinds of microplate readers and plate types. A journal article on this work was submitted this year.