Escherichia coli as a model active colloid: A practical introduction

Abstract

The flagellated bacterium Escherichia coli is increasingly used experimentally as a self-propelled swimmer. To obtain meaningful, quantitative results that are comparable between different laboratories, reproducible protocols are needed to control, 'tune' and monitor the swimming behaviour of these motile cells. We critically review the knowledge needed to do so, explain methods for characterising the colloidal and motile properties of E. coli cells, and propose a protocol for keeping them swimming at constant speed at finite bulk concentrations. In the process of establishing this protocol, we use motility as a high-throughput probe of aspects of cellular physiology via the coupling between swimming speed and the proton motive force. Keywords:Escherichia coli, active colloids, motility, differential dynamic microscopy, metabolism, bioenergetics, proton motive force Some time ago, our lab wanted to culture motile bacteria as 'model active colloids'. We obtained a strain of Escherichia coli with the full complement of motility genes and a culturing protocol from a local microbiologist. For some time, we thought we were experimenting with motile E. coli, until one day we checked in the microscope. Few, if any, of the cells were swimming! So we set out to learn how to modify the standard protocol to optimise motility by collating literature, talking to other researchers and trial and error; we also implemented differential dynamic microscopy (DDM) to quantify motility.This article reviews what we have learnt. Some of the material is previously known, but seldom critically discussed in one place. We have explained some basic bacterial bioenergetics and genetics, because physical scientists can use E. coli and collaborate with biologists more effectively if these topics are understood. Much of the materials is new, arising from using DDM to quantify motility. While we aim primarily at researchers working on active colloids [1], this article should also be useful to others studying motility biophysics [2].From the outset, we refer to various culture media (BMB, TB, LB) and protocols, and freely use terminology related to molecular biology (plasmid, gene names, etc.) and measurement techniques (OD, DDM, etc.). Readers should refer to Section 5 on matters of cell culture, Sections 3 and 4 for methodology, and Section 10 and Appendix C for biological jargon. We also provide a table of symbols in Appendix F.

Osmotaxis in Escherichia coli through changes in motor speed

“…In DDM experiments, the mean swimming speed was calculated from DDM movies as an average over ∼10 4 cells/ml. Details of the image processing and data analysis were as before [Wilson et al, 2011,Martinez et al, 2012,Martinez et al, 2014,Schwarz-Linek et al, 2016. DDM allows the measurement of an advective speed, simultaneously, over a range of spatialfrequency q, where each q defines a length-scale L=2*pi/q (for more details, see [Wilson et al, 2011, Martinez et al, 2012).…”

“…3D for all magnitudes of osmotic upshocks was previously characterized in Motility Buffer, where E. coli maintains Proton Motive Force (PMF) using endogenous energy sources [Dawes andRibbons, 1965, Schwarz-Linek et al, 2016]. At fixed buffer composition, the time it takes to consume all available oxygen is inversely proportional to the cell concentration, and upon oxygen exhaustion a sudden 'crash' in swimming speed occurs [Schwarz-Linek et al, 2016]. Interestingly, we do not see such a 'crash' in swimming speed for the lowest five values of the imposed upshock, but do see a 'crash' when the upshock is at the highest value of 785 mOsmol/kg.…”

“…After washing, all the experiments were performed in VR buffer. KAF95 and KAF84 cells were washed by centrifuging them into a pellet and exchanging solution, while MG1655 cells were washed by gentle filtration to preserve filaments [Schwarz-Linek et al, 2016] and experiments were performed in VR buffer as for motor speed measurements.…”

Dynamics of ellipsoidal tracers in swimming algal suspensions

“…The self-propelled particles can convert ambient or internal free energy into persistent motions with a direction depending on the local configuration of particles and interparticle interactions. Suspensions of swimming microorganisms and synthetic colloidal swimmers are the most widely studied active fluids in experiments and frequently serve as models for theoretical investigations [4,6]. Joint efforts of experiments, simulations, and theories have shown that active fluids exhibit surprising behaviors such as giant number fluctuations [7][8][9], ordered phases with collective particle motions [3,10,11], and abnormal rheology [12][13][14][15], unknown to conventional equilibrium complex fluids.…”

“…Active fluids are a novel class of nonequilibrium soft materials, which are composed of a large number of selfpropelled particles suspended in simple fluids [1][2][3][4][5]. The self-propelled particles can convert ambient or internal free energy into persistent motions with a direction depending on the local configuration of particles and interparticle interactions.…”