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). In order to fully exploit this technology, we need methods to rapidly construct reporter genes, both on plasmids and on the chromosome, mathematical models to infer biologically relevant quantities from the primary data, and computer tools to achieve this in an efficient and user-friendly manner. For instance, in a typical microplate experiment, 96 cultures are followed in parallel, over several hours, resulting in 10,000-100,000 measurements of absorbance and fluorescence and luminescence intensities.

Valentin Zulkower, former PhD student in IBIS, 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 in the analysis of reporter gene data, notably the inference of growth rate, promoter activity, and protein concentration profiles, to be solved in a mathematically sound and practical manner. The linear inversion methods, published in Bioinformatics in 2015 [12], in 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), Yannick Martin has 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 web service has been deployed on the IFB cloud and on an Inria server, accompanied by extensive user documentation, online help, and a tutorial. A paper on WellInverter is in preparation.

While the use of microplate readers results in population-level measurements of gene expression, for many applications it is mandatory to monitor gene expression over time on the level of individual cells. Several developments in the past decade have enormously extended the capabilities to achieve this, in particular the combination of fluorescence time-lapse microscopy for precisely quantifying gene expression in single cells and microfluidics technology for cultivating bacteria in confined spatial compartments and under well-controlled experimental conditions. One of the most wide-spread microfluidics devices is the so-called mother machine shown in Figure 5. A major problem is that functional software for image analysis (segmentation, tracking, lineage reconstruction, ...) adapted to the requirements of mother machine applications are still rare. IBIS has therefore collaborated with the BEAGLE project-team for the adaptation of their tool FluoBacTracker to the analysis of time-lapse movies of fluorescent reporter expression and bacterial growth in microfluidics devices. This collaboration, which has also involved the SERPICO project-team, was supported by the Technology Transfer and Innovation department of Inria, in the framework of the Inria Hub program, and has allowed the hiring of Cyril Dutrieux as a software engineer in IBIS.