bioRxiv Science⌕ Search

Biology subjects

Okumus, B.

Publications and source records attributed to Okumus, B..

2 recordsLinked to original sources

Dynamic Regulation of Growth and Physiology of Microbes under Complex Changing Conditions

As bacteria transition from exponential to stationary phase they change greatly in size, morphology, growth and expression-profiles. These responses also vary between individual cells, but it has proven difficult to track cell lineages along the growth curve to determine the progression of events or correlations between how individual cells enter and exit dormancy. We developed a platform for tracking >105 parallel cell lineages in dense and changing cultures, independently validating that the imaged cells closely track batch populations. Initial applications show that for both Escherichia coli and Bacillus subtilis, growth changes from an adder mode in exponential phase to mixed adders-timers entering stationary phase, and then a near-perfect sizer upon exit - creating broadly distributed cell sizes in stationary phase but rapidly returning to narrowly distributed sizes upon exit. Furthermore, cells that undergo more divisions entering stationary phase suffer reduced survival after long periods of dormancy but are the only cells observed that persist against antibiotics.

microbiology↗

Quantification of very low-abundant proteins in bacteria using the HaloTag and epi-fluorescence microscopy

Cell biology is increasingly dependent on quantitative methods resulting in the need for microscopic labelling technologies that are highly sensitive and specific. Whilst the use of fluorescent proteins has led to major advances, they also suffer from their relatively low brightness and photo-stability, making the detection of very low abundance proteins using fluorescent protein-based methods challenging. Here, we characterize the use of the self-labelling protein tag called HaloTag, in conjunction with an organic fluorescent dye, to label and accurately count endogenous proteins present in very low numbers (<7) in individual Escherichia coli cells. This procedure can be used to detect single molecules in fixed cells with conventional epifluorescence illumination and a standard microscope. We show that the detection efficiency of proteins labelled with the HaloTag is [&ge;]80%, which is on par or better than previous techniques. Therefore, this method offers a simple and attractive alternative to current procedures to detect low abundance molecules.

systems biology↗