Protocols in Current Issue
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0 Q&A 3667 Views Jul 5, 2020
The natural environment of microbial cells like bacteria and yeast is often a complex community in which growth and internal organization reflect morphogenetic processes and interactions that are dependent on spatial position and time. While most of research is performed in simple homogeneous environments (e.g., bulk liquid cultures), which cannot capture full spatiotemporal community dynamics, studying biofilms or colonies is complex and usually does not give access to the spatiotemporal dynamics at single cell level. Here, we detail a protocol for generation of a microfluidic device, the “yeast machine”, with arrays of long monolayers of yeast colonies to advance the global understanding of how intercellular metabolic interactions affect the internal structure of colonies within defined and customizable spatial dimensions. With Saccharomyces cerevisiae as a model yeast system we used the “yeast machine” to demonstrate the emergence of glucose gradients by following expression of fluorescently labelled hexose transporters. We further quantified the expression spatial patterns with intra-colony growth rates and expression of other genes regulated by glucose availability. In addition to this, we showed that gradients of amino acids also form within a colony, potentially opening similar approaches to study spatiotemporal formation of gradients of many other nutrients and metabolic waste products. This approach could be used in the future to decipher the interplay between long-range metabolic interactions, cellular development, and morphogenesis in other same species or more complex multi-species systems at single-cell resolution and timescales relevant to ecology and evolution.
0 Q&A 3805 Views May 20, 2019
Biofilms are bacterial communities in the shape of exopolysaccharide matrix-encased aggregates attached onto interphases able to resist environmental aggressions. The development of bacteria in the shape of biofilms deeply affects the performance of many industrial processes which work with fluidic systems, where bacteria may settle and prosper. As a consequence industrial equipment experiments low performance issues and substantial maintenance costs.

The study of how bacteria of industrial interest such as Pseudomonas putida spread in these fluidic systems is highly dependent on the chosen experimental system to retrieve such data, thus using scaled prototypes becomes an essential step towards the design of a more efficient system to handle biofilms, either to control them or to prevent them. This protocol describes how to assemble, operate and maintain a device to grow and monitor the biofilm spreading pattern of this bacterium (as a function of the fluid hydrodynamics) in a custom-made chamber larger than those typically used in laboratory environments, and how to analyze the information gathered from it in a straightforward fashion. Description of the protocol was thought to be used as a working template not only for the presented case study but for any other potential experiment in different contexts and diverse scales following similar design principles.
0 Q&A 6479 Views Aug 20, 2018
Bacteria in nature live in complex communities with multiple cell types and spatially-dependent interactions. Studying cells in well-mixed environments such as shaking culture tubes or flasks cannot capture these spatial dynamics, but cells growing in full-fledged biofilms are difficult to observe in real time. We present here a protocol for observing time-resolved, multi-species interactions at single-cell resolution. The protocol involves growing bacterial cells in a near monolayer in a microfluidic device. As a demonstration, we describe in particular observing the dynamic interactions between E. coli and Acinetobacter baylyi. In this case, the protocol is capable of observing both contact-dependent lysis of E. coli by A. baylyi via the Type VI Secretion System (T6SS) and subsequent functional horizontal gene transfer (HGT) of genes from E. coli to A. baylyi.

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