Available online at www.sciencedirect.com
Flow cytometry for bacteria: enabling metabolic engineering,
synthetic biology and the elucidation of complex phenotypes
Bryan P Tracy
1
, Stefan M Gaida
2
and Eleftherios T Papoutsakis
2
Flow cytometry (FC) and FC-based cell sorting have been
established as critical tools in modern cell and developmental
biology. Yet, their applications in bacteria, especially in the
multiparametric mode, remain limited. We argue that FC
technologies have the potential to greatly accelerate the
analysis and development of microbial complex phenotypes
through applications of metabolic engineering, synthetic
biology, and evolutionary engineering. We demonstrate the
importance of FC for elucidating population heterogeneity
because of developmental processes or epigenetic regulation.
FC can be engaged for both synthetic and analytical
applications of complex phenotypes within a single species,
multispecies, and microbial-library populations. Examples
include methods to identify developmental microbial stages
associated with productive metabolic phenotypes, select
desirable promoters from a single species or metagenomic
libraries, and to screen designer riboswitches for synthetic-
biology applications.
Addresses
1
Elcriton, Inc., Delaware Biotechnology Institute, Rm 288, 15 Innovation
Way, Newark, DE 19711, USA
2
Department of Chemical Engineering & The Delaware Biotechnology
Institute, University of Delaware, 15 Innovation Way, Newark, DE 19711,
USA
Corresponding author: Papoutsakis, Eleftherios T (epaps@udel.edu)
Current Opinion in Biotechnology 2010, 21:85–99
This review comes from a themed issue on
Analytical biotechnology
Edited by Peter Neubauer and Andreas Schmid
0958-1669/$ – see front matter
# 2010 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2010.02.006
Introduction
What is flow cytometry (FC): to measure the detailed
properties of individuals in a population, and sort the
individuals according to desired properties
Flow cytometry (FC) is a flow-based method that enables
the simultaneous measurement of multiple physical and
chemical/biological characteristics of single particles,
which typically are cells. On the basis of the principle
of hydrodynamic focusing, the flow cytometer fluidics
system transports particles in a fluid stream, one cell at a
time at high speed, to a laser beam for the interrogation of
particle properties. At the laser intercept, one or multiple
laser beams illuminate the cells in order to measure their
light scattering properties and to excite fluorescent mol-
ecules. Suitable lasers can excite naturally fluorescent
cellular molecules and fluorescent molecules specifically
employed to tag or probe (stain) various cellular com-
ponents or processes. The optics system also includes
lenses, beam splitters, and filters which are used to direct
the incident light scattered by the cells, or the emitted
fluorescent light that results from the cell illumination, to
light detectors. Light signals captured by these detectors
are converted into electronic signals by the cytometer’s
electronic system, which then filters and processes these
signals using computer-based algorithms. Cell/particle
properties measured include a particle’s relative size,
relative granularity or internal complexity, and relative
fluorescence intensity of the specific fluorescent mol-
ecule(s) being interrogated. Fluorescent dyes can be used
to directly or indirectly (e.g. using antibodies) label
cellular components such as DNA, surface proteins/
receptors, intracellular structural proteins and enzymes,
specific nucleic acids (NAs), membrane properties and
ion fluxes, secreted proteins or small molecules, and cell
organelles. While not all of these have been widely
applied in bacteria, the potential of such applications is
becoming increasingly apparent.
The flow cytometer can be also equipped with a cell-
sorting capability for Fluorescence-Activated Cell Sorting
(FACS) analysis. While the term FACS is a registered
trademark of the Becton-Dickinson Company, it is widely
used in a generic way by the scientific community. Sorting
can be based on different methods of capturing the cells,
and sorting decisions can be based on one or multiple
cellular properties. Cells (single, a few, or in thousands to
millions) thus collected in tubes or wells can be used for
further assays, microscopy, and molecular and functional
assays. If the staining method allows, cells can be cultured
for further assays or enrichment.
FC and FACS: from applications in cell and
developmental biology and medicine to
microbial systems
Applications of FC and FACS are abundant for identify-
ing and sorting cells with desirable properties for basic
studies in cell and developmental biology, as well clinical
applications [1,2]. These applications have facilitated the
development of FC/FACS instruments, fluorescent
probes, and methods to label cells, analyze the fluorescent
signals and sort cells to achieve a desirable analytical or
clinical outcome. However, this technology still remains
www.sciencedirect.com Current Opinion in Biotechnology 2010, 21:85–99