Gene machines

Abstract:

Dataset of microscopy images of untreated and treated E.coli lab strains and clinical isolates, and machine learning models trained on them. Corresponding publications: https://doi.org/10.1101/2022.12.08.22283219 Corresponding analysis code: https://github.com/KapanidisLab/Deep-Learning-and-Single-Cell-Phenotyping-for-Rapid-Antimicrobial-Susceptibility-Testing

Abstract:

ABSTRACT

The increasing frequency and magnitude of viral outbreaks in recent decades, epitomized by the current COVID-19 pandemic, has resulted in an urgent need for rapid and sensitive diagnostic methods. Here, we present a methodology for virus detection and identification that uses a convolutional neural network to distinguish between microscopy images of single intact particles of different viruses. Our assay achieves labeling, imaging and virus identification in less than five minutes and does not require any lysis, purification or amplification steps. The trained neural network was able to differentiate SARS-CoV-2 from negative clinical samples, as well as from other common respiratory pathogens such as influenza and seasonal human coronaviruses. Additionally, we were able to differentiate closely related strains of influenza, as well as SARS-CoV-2 variants. Single-particle imaging combined with deep learning therefore offers a promising alternative to traditional viral diagnostic and genomic sequencing methods, and has the potential for significant impact.

Abstract:

Structured Illumination Microscopy, SIM, is one of the most powerful optical imaging methods available to visualize biological environments at subcellular resolution. Its limitations stem from a difficulty of imaging in multiple color channels at once, which reduces imaging speed. Furthermore, there is substantial experimental complexity in setting up SIM systems, preventing a widespread adoption. Here, we present Machine-learning Assisted, Interferometric Structured Illumination Microscopy, MAI-SIM, as an easy-to-implement method for live cell super-resolution imaging at high speed and in multiple colors. The instrument is based on an interferometer design in which illumination patterns are generated, rotated, and stepped in phase through movement of a single galvanometric mirror element. The design is robust, flexible, and works for all wavelengths. We complement the unique properties of the microscope with an open source machine-learning toolbox that permits real-time reconstructions to be performed, providing instant visualization of super-resolved images from live biological samples.

Abstract:

Rho is a general transcription termination factor in bacteria, but many aspects of its mechanism of action are unclear. Diverse models have been proposed for the initial interaction between the RNA polymerase (RNAP) and Rho (catch-up and stand-by pre-terminational models); for the terminational release of the RNA transcript (RNA shearing, RNAP hyper-translocation or displacing, and allosteric models); and for the post-terminational outcome (whether the RNAP dissociates or remains bound to the DNA). Here, we use single-molecule fluorescence assays to study those three steps in transcription termination mediated by E. coli Rho. We find that different mechanisms previously proposed for each step co-exist, but apparently occur on various timescales and tend to lead to specific outcomes. Our results indicate that three kinetically distinct routes take place: (1) the catch-up mode leads first to RNA shearing for RNAP recycling on DNA, and (2) later to RNAP displacement for decomposition of the transcriptional complex; (3) the last termination usually follows the stand-by mode with displacing for decomposing. This three-route model would help reconcile current controversies on the mechanisms.

Abstract:

The expression of most bacterial genes commences with the binding of RNA polymerase (RNAP)-σ⁷⁰ holoenzyme to the promoter DNA. This initial RNAP-promoter closed complex undergoes a series of conformational changes, including the formation of a transcription bubble on the promoter and the loading of template DNA strand into the RNAP active site; these changes lead to the catalytically active open complex (RP_O) state. Recent cryo-electron microscopy studies have provided detailed structural insight on the RP_O and putative intermediates on its formation pathway. Here, we employ single-molecule fluorescence microscopy to interrogate the conformational dynamics and reaction kinetics during real-time RP_O formation on a consensus lac promoter. We find that the promoter opening may proceed rapidly from the closed to open conformation in a single apparent step, or may instead involve a significant intermediate between these states. The formed RP_O complexes are also different with respect to their transcription bubble stability. The RNAP cleft loops, and especially the β' rudder, stabilise the transcription bubble. The RNAP interactions with the promoter upstream sequence (beyond -35) stimulate transcription bubble nucleation and tune the reaction path towards stable forms of the RP_O.