Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

Abstract

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

A Microfluidics and Agent-Based Modeling Framework for Investigating Spatial Organization in Bacterial Colonies: The Case of Pseudomonas Aeruginosa and H1-Type VI Secretion Interactions

“…aeruginosa PAO1 mutants were modeled during growth simulations to investigate the effects of Type VI secretion on cell organization. Cultures included a ΔretS mutant that constitutively expresses GFP and the toxic effector proteins associated with Type VI secretion, and a ΔretS/Δtse/i1-6 deletion mutant that constitutively expresses m-Cherry and is susceptible to Type VI secretion interactions (i.e., injection of toxic effector proteins) (Timm et al, 2017 ). Cell culture conditions for growth experiments followed Hood et al ( 2010 ) and Timm et al ( 2017 ).…”

“…The cell-seeding, growth, imaging and image analysis methods used in this study correspond to those described in Timm et al ( 2017 ). Timm et al ( 2017 ) provides a detailed step-by-step description of those protocols along with online video content displaying those techniques. Representative data, as well as the image collection and correction procedures, are provided there.…”

“…Representative data, as well as the image collection and correction procedures, are provided there. Specifically, we provide additional analysis of a more comprehensive data set and describe the development of a new ABM framework that helps explain some of the unresolved questions reported by Timm et al ( 2017 ). Briefly, cells of P .…”

“…Spatial organization has a strong influence on the development and dynamics of biological systems (Kreft et al, 1998 ; Lardon et al, 2011 ; Halsted et al, 2016 ; Hansen et al, 2016 ; McNally et al, 2017 ; Timm et al, 2017 ). The factors leading to changes in organization of multicellular assemblages at fine spatial scales are not well characterized or understood, however, they are expected to guide the succession of community development and function toward specific outcomes (Liu et al, 2009 ; Cline and Zak, 2015 ; Dini-Andreote et al, 2015 ).…”

“…Numerous advances in our understanding of cell-to-cell behavior and interactions at fine spatial scales have stemmed from the use and development of nano/micro-fabricated platforms (Wang et al, 2013 ; Yamazaki et al, 2014 ; Swennenhuis et al, 2015 ; Xue et al, 2015 ; Hansen et al, 2016 ; Zhang et al, 2016 ; Timm et al, 2017 ; Yeh et al, 2017 ). Timm et al ( 2017 ) used a microwell array platform to study the contact-mediated T6SS interactions of P . aeruginosa .…”