PROJECT PROPOSAL
for applicants for Ph.D. fellowships
supervisor:
institution:
contact:
CV:
Péter GALAJDA*, Ph.D.
Institute of Biophysics
galajda.peter@brc.mta.hu
http://www.brc.mta.hu/file/cv/bf_galajda_peter_en.pdf
project title:
STUDYING AGING AND PHENOTYPIC HETEROGENEITY
OF BACTERIA USING HOLOGRAPHIC OPTICAL
TWEEZERS
PROJECT SUMMARY
Bacteria reproduce by binary fission after which two seemingly identical daughter cells are
produced. This mode of reproduction raises several questions. Without a clear distinction between
“parents” and “daughters”, can the concept of “age” applied to bacteria? Are the daughter cells really
identical/similar (phenotypically)? How does (phenotypic) variability emerge in a clonal bacterial
population?
During the project we use a novel experimental platform based on optical trapping and microfluidics
to study a growing colony with single cell level of detail for an extended period of time. We track cell
elongation and division rate, cell survival, expression levels of proteins and swimming behavior in a
constantly dividing population of E. coli bacteria. We expect to see how variability and heterogeneity
of these measured parameters develop in a clonal population descended from a single cell.
BACKGROUND OF THE STUDY
The cellular reproduction and the cell cycle are
fundamental processes of life. Thus it is
important to understand the mechanisms
underlying these processes for bacteria.
Although asexual reproduction of bacterial cells
results in genetically identical daughter cells,
phenotypic differences may emerge. These
differences lead to a heterogeneity even in a
clonal population. That variety ultimately may
be a key for the survival of the population.
Phenotypic variability even in a clonal
population may be an important factor in
bacterial infections. A potential treatment have
to consider the heterogeneity of the infecting
population. Therefore understanding the
mechanisms behind the emergence of
heterogeneity may help us preventing or
treating such infections.
Our novel approach eliminates the limitation of
previous experimental methods in the field. We
combine leading technologies of holographic
optical manipulation, microfluidics and optical
microscopy. The experimental platform offers:
- Single cell level measurements (instead of
measuring ensemble averages)
- Tracking cell lineage for each single cell in the
sample
- Large number (up to tens of thousands) of
cells may be measured in a single experiment
- Long timescales are accessible: cells may be
tracked through hundreds of generations
- Manipulation capabilities for each single cell in
the sample: cells may be trapped, moved or
released under full control. This enables the
selection of the cells to be measured analyzed.
RELEVANT RESEARCH IN THE HOST
LABORATORY
Our research group uses microfluidic methods
to study various biological and biophysical
phenomena related to bacterial populations and
communities. We aim to understand principles
governing the formation and development of
bacterial communities on various level of
organization ranging from single cells to
multicellular multispecies bacterial societies.
Within this framework we study both cellenvironment and cell-cell interactions. In our
reserach
we
employ
microfabrication
techniques to create engineered artificial
habitats that, when populated with bacteria, are
suitable to investigate the above problems.
Previously we focused to population level
phenomena. We studied the hydrodynamics of
bacterial swimming. We have shown using
microfluidic structures that population level
coordinated motion patterns develop when cell
densities are high. These patterns shaped by
the geometry of physical boundaries. This
phenomenon offers a way to manipulate
swimming bacterial populations using artificial
obstacles.
We have developed a microfluidic device to
study bacterial chemotaxis. Using this we have
tested the chemoeffector potential of various
chemicals that play a role in the organization of
bacterial communities. We have shown for
example that the communication signaling
molecules of the bacterium P. aeruginosa
induce a chemotactic response in E. coli. We
have observed similar effects for some
secondary metabolites. We currently investigate
the effect of various antibiotics on bacterial
chemotaxis. We also study how the uneven
spatial distribution of antibiotics affects the
evolution of resistance.
The device we developed is also suitable for
the observation of chemically coupled but
physically separated bacterial populations. We
can show the complex interaction between
populations of the same strain but also between
different strains, and at the same time we aim
to identify the chemicals through which these
interactions act.
Our microfluidic devices enable us to study how
the physical structure of the habitat affects the
competition of different bacterial strains. We
have shown, for example, that fragmented,
patchy habitats support the survival of
cooperating bacteria against “selfish” non
cooperating ones.
Supported by the
TÁMOP 4.1.1.C -13/1/KONV.2014-0001
project
In our previous works we focused on population
level phenomena, next we aim to explore the
single cell level details of the observed
phenomena. It has a great importance that
individuals are not exactly the same in a
population even in case of an identical genetic
background. We would like to better understand
the role of this phenotypic heterogeneity as well
as its role in the hierarchical transition between
individuals and communities.
SPECIFIC AIMS
Our research plan aims to develop a new
experimental platform and apply it to answer
fundamental biological questions related to
bacterial reproduction, cell cycle, aging and
phenotypic variability.
The core biological question we ask is: how
bacterial cells of successive generations differ
from their ancestors?
Previous studies suggest that various cellular
characteristics such as growth rate and survival
probability might slightly change after cell
division. However these studies suffer from
several limitations, and most often are based on
a limited amount of data. Our experimental
method overcomes these limitations: we can
collect large amounts of data (in the order of
10000 cells per experiment), but we have full
manipulative control over each single cell. As a
consequence a wide variety of questions may
be studied.
The specific questions we study are the
following.
How does phenotypic variability emerge in a
clonal population? It is an important question
since phenotypic heterogeneity contributes to
the survival probability of a population. The
measured cellular parameters include cell size,
elongation rate, division time (and frequency),
protein expression rate (based on fluorescent
labeling), and motility (tumbling rate).
How does the division rate and the survival
probability change after several hundred
generations? Can we explain the observations
based on a novel concept of bacterial age?
In order to answer these questions we develop
an experimental platform based on microfluidics
and optical micromanipulation. The microfluidic
components make it possible to precisely
control the culturing conditions, while optical
manipulation helps us to follow the cell lineage
relations and select the cells to analyze. We
plan to put together an extensive database
where culturing conditions, cell lineage
informations, cell pole age and various
phenotypic characteristics (determined by
optical microscopy) are included for tens of
thousands of cells and hundreds of
generations.
MATERIAL AND METHODS
 Basic methods in microbiology, cell culturing,
transformation, analysis of cells and cultures
 Computer aided design of microfluidic
systems
 Production of microfluidic systems by various
microfabrication
techniques,
including,
photolithography and soft-lithography
 Optical micromanipulation: using and
development of a laser tweezers system
 Use of light microscopy (transmission,
fluorescence) techniques on biological
samples
 Computer based image processing and
analysis, programming tasks
SUGGESTED READINGS
Cookson S, et al.: Monitoring Dynamics of Single-Cell
Gene Expression Over Multiple Cell Cycles. Mol. Syst.
Biol., 1:2005.0024(2005)
Grier DG, et al.: Holographic Optical Trapping. Appl. Opt.,
45:880–887(2006)
Pin C, et al.: Single-Cell and Population Lag Times as a
Function of Cell Age. Appl. Environ. Microb., 74:25342536(2008)
Stewart EJ, et al.: Aging and Death in an Organism That
Reproduces by Morphologically Symmetric Division. Plos
Biol., 3:e45(2005)
Strovas TJ, et al.: Cell-to-Cell Heterogeneity in Growth
Rate and Gene Expression in Methylobacterium
Extorquens AM1. J. Bacteriol., 189:7127-7133(2007)
Wakamoto Y, et al.: Analysis of Single-Cell Differences
by Use of an on-Chip Microculture System and Optical
Trapping. Fresen. J. Anal. Chem., 371:276-281(2001)
Wang P, et al.: Robust Growth of Escherichia Coli. Curr.
Biol., 20:1099-1103(2010)
Zhang H, et al.: Optical Tweezers for Single Cells. J. Roy.
Soc. Interface, 5:671-690(2008)
SNAPSHOTS FROM THE HOST LABORATORY
Significant publications
Nagy K, et al.: Interaction of Bacterial Populations in Coupled Microchambers. Chem. Biochem. Eng. Q., 28:225-231(2014)
Hol FJH, et al.: Spatial Structure Facilitates Cooperation in a Social Dilemma: Empirical Evidence from a Bacterial
Community. PLoS One, 8:e77042(2013)
Keymer JE, et al.: Computation of mutual fitness by competing bacteria. P. Natl. Acad. Sci. USA, 105:20269-20273(2008)
Galajda P, et al.: A wall of funnels concentrates swimming bacteria. J. Bacteriol., 189:8704-8707(2007)
Galajda P, et al.: Complex micromachines produced and driven by light
Appl. Phys. Lett., 78:249-251(2001)
Representative recent research grants
“Lendület” (Hung. Acad. Sci., 2010-)
Some of the latest students in the laboratory
Hodula O, Ph.D., 2013-recent; “Studying bacterial chemotaxis using microfluidic methods”
Sipos O, Ph.D., 2010-recent; “Studying bacterial swimming motility using microfluidic methods”
Elek R, B.Sc., 2014, “Development of a hydrogel based microfluidic device to create chemical gradients”
Balog JÁ, B.Sc., 2014, “The effects of antibiotics on the swimming motility of bacteria”
Dér L, B.Sc., 2014, “Development of a computer controlled active microfluidics system”
Supported by the
TÁMOP 4.1.1.C -13/1/KONV.2014-0001
project