Microdroplet-enabled highly parallel co-cultivation of microbial communities.
ABSTRACT Microbial interactions in natural microbiota are, in many cases, crucial for the sustenance of the communities, but the precise nature of these interactions remain largely unknown because of the inherent complexity and difficulties in laboratory cultivation. Conventional pure culture-oriented cultivation does not account for these interactions mediated by small molecules, which severely limits its utility in cultivating and studying "unculturable" microorganisms from synergistic communities. In this study, we developed a simple microfluidic device for highly parallel co-cultivation of symbiotic microbial communities and demonstrated its effectiveness in discovering synergistic interactions among microbes. Using aqueous micro-droplets dispersed in a continuous oil phase, the device could readily encapsulate and co-cultivate subsets of a community. A large number of droplets, up to ∼1,400 in a 10 mm × 5 mm chamber, were generated with a frequency of 500 droplets/sec. A synthetic model system consisting of cross-feeding E. coli mutants was used to mimic compositions of symbionts and other microbes in natural microbial communities. Our device was able to detect a pair-wise symbiotic relationship when one partner accounted for as low as 1% of the total population or each symbiont was about 3% of the artificial community.
- [Show abstract] [Hide abstract]
ABSTRACT: The oceans harbor a tremendous diversity of marine microbes. Different functional groups of bacteria, archaea, and protists arise from this diversity to dominate various habitats and drive globally important biogeochemical cycles. Explanations for the distribution of microbial taxa and their associated activity often focus on resource availability and abiotic conditions. However, the continual reshaping of communities by mortality, allelopathy, symbiosis, and other processes shows that community interactions exert strong selective pressure on marine microbes. Deeper exploration of microbial interactions is now possible via molecular prospecting and taxon-specific experimental approaches. A holistic outlook that encompasses the full array of selective pressures on individuals will help elucidate the maintenance of microbial diversity and the regulation of biogeochemical reactions by planktonic communities.Science 06/2008; 320(5879):1043-5. · 31.48 Impact Factor
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ABSTRACT: Soil microbial communities are integrally involved in biogeochemical cycles and their activities are crucial to the productivity of terrestrial ecosystems. Despite the importance of soil microorganisms, little is known about the distribution of microorganisms in the soil or the manner in which microbial community structure responds to changes in land management. We investigated the structure of microbial communities in the soil over two years in a series of replicated plots, that included, cultivated fields, fields abandoned from cultivation and fields with no history of cultivation. Microbial community structure was examined by monitoring the relative abundance of ribosomal RNA (rRNA) from seven of the most common bacterial groups in soil (the Alpha and Beta Proteobacteria, Actinobacteria, Cytophagales, Planctomycetes, Verrucomicrobia and the Acidobacteria) and the Eukarya. These data reveal that soil microbial communities are dynamic, capable of significant change at temporal scales relative to seasonal events. However, despite temporal change in microbial community structure, the rRNA relative abundance of particular microbial groups is affected by the local environment such that recognizable patterns of community structure exist in relation to field management.Environmental Microbiology 07/2003; 5(6):441-52. · 6.24 Impact Factor
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ABSTRACT: It is becoming increasingly difficult to isolate novel useful microorganisms from the natural environment using conventional screening methods based on pure culture techniques. A novel method for screening microorganisms in symbiotic association with insects was developed. This method involves the following two steps. In the first step, the existence of desired microorganisms that grow well by degrading difficult-to-degrade materials in the gut of insects is detected using the survivability of insects as an indicator. In the second step, the desired microorganisms are selected from the surviving insects. The second step is based on an idea that the guts of insects act as continuous-culture systems whereby microorganisms that cannot degrade diet components are washed out whereas those that can degrade diet components are retained and made to multiply in the gut. Coptotermes formosanus Shiraki was fed with an artificial diet containing phenol as a model of lignin-derived and difficult-to-degrade compound. Each C. formosanus feeding on an artificial diet containing 100 mg/l phenol had different levels of adaptation to the toxicity of phenol. About 20% of C. formosanus fed with an artificial diet containing 100 mg/l phenol died within a few days whereas others survived for more than 10 d. The structure of the intestinal microorganisms of the surviving C. formosanus fed with the 100 mg/l phenol artificial diet gradually changed and was very different from that of the bacterial communities obtained from the enrichment culture of wood-feeding C. formosanus using an artificial medium containing phenol as a sole carbon source. Furthermore, Only three species (as DGGE band) were detected from the gut of wood-feeding C. formosanus, whereas 200 times more phenol-degrading microorganisms were detected in the gut of C. formosanus feeding on a phenol artificial diet. Out of these nine species (as DGGE band) of phenol-degrading microorganisms were isolated. The screening method developed in this study can also be applied to various insects, leading to the isolation of various microorganisms that can degrade difficult-to-degrade compounds.Journal of Bioscience and Bioengineering 05/2007; 103(4):358-67. · 1.79 Impact Factor
Microdroplet-Enabled Highly Parallel Co-Cultivation of
Jihyang Park1, Alissa Kerner2, Mark A. Burns1,2, Xiaoxia Nina Lin1,2,3*
1Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America, 2Department of Biomedical Engineering, University of
Michigan, Ann Arbor, Michigan, United States of America, 3Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, United
States of America
Microbial interactions in natural microbiota are, in many cases, crucial for the sustenance of the communities, but the
precise nature of these interactions remain largely unknown because of the inherent complexity and difficulties in
laboratory cultivation. Conventional pure culture-oriented cultivation does not account for these interactions mediated by
small molecules, which severely limits its utility in cultivating and studying ‘‘unculturable’’ microorganisms from synergistic
communities. In this study, we developed a simple microfluidic device for highly parallel co-cultivation of symbiotic
microbial communities and demonstrated its effectiveness in discovering synergistic interactions among microbes. Using
aqueous micro-droplets dispersed in a continuous oil phase, the device could readily encapsulate and co-cultivate subsets
of a community. A large number of droplets, up to ,1,400 in a 10 mm65 mm chamber, were generated with a frequency
of 500 droplets/sec. A synthetic model system consisting of cross-feeding E. coli mutants was used to mimic compositions
of symbionts and other microbes in natural microbial communities. Our device was able to detect a pair-wise symbiotic
relationship when one partner accounted for as low as 1% of the total population or each symbiont was about 3% of the
Citation: Park J, Kerner A, Burns MA, Lin XN (2011) Microdroplet-Enabled Highly Parallel Co-Cultivation of Microbial Communities. PLoS ONE 6(2): e17019.
Editor: Alfredo Herrera-Estrella, Cinvestav, Mexico
Received September 28, 2010; Accepted January 18, 2011; Published February 25, 2011
Copyright: ? 2011 Park et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by an Elizabeth C. Crosby Research Award received by XNL, an NIH grant (1R01EB006789) received by MAB, and an NIH grant
(1R21HG005077) received by XNL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: RainDance Technologies provided the PFPE-PEG block copolymer surfactant used in this work and permitted its use solely for the
purpose of scientific research at the University of Michigan only under the direction of Dr. Xiaoxia Lin. This does not alter the authors’ adherence to all the PLoS
ONE policies on sharing data and materials.
* E-mail: firstname.lastname@example.org
In nature, most microbes live in synergistic communities as a
way to adapt to and thrive in various environments, such as the
ocean[1,2], soil[3,4], and higher organisms as hosts[5,6]. These
microbial communities play important roles in a wide spectrum of
ecosystems and form diverse interactions among community
members and with their surroundings. For example, the human
body is a representative host for natural microbial communities:
over 100 trillion bacteria are estimated to be present in the human
gut, more than 600 microbial species are known to inhabit the
human oral cavity, and over 100 different bacterial 16S rRNA
are present on human skin. These microbes are believed to be
closely related to human health. For instance, the gut
microbiota is known to contribute to digestion of nutrients,
stimulation of immunity and protection of the host from
inflammatory diseases. Despite their ubiquitousness and
apparent significance, our understanding of these microbial
communities remains very limited, largely owing to their inherent
complexity and the difficulty in laboratory cultivation of most of
The majority of existing microbial species, estimated to be in
the millions, have not been cultured in the laboratory,
which severely limits the extent to which they can be charac-
terized and further studied. One important reason behind this
‘‘unculturability’’ is that conventional laboratory cultivation is
aimed at pure cultures of individual species, while in nature, the
survival and growth of microorganisms are largely associated with
their interactions with other members of the community they live
in[7,16,17,18]. These interactions are mediated by various
molecules such as secondary metabolites, quorum sensing
molecules, and peptides[19,20,21]. Accordingly, researchers have
attempted to develop alternative cultivation techniques that allow
interactions among microbes[16,22,23,24,25].
Kaeberlein et al. successfully isolated and cultured previously
uncultivated marine microorganisms by using a multi-chamber
set-up which allowed the diffusion of small molecules through
Recent years have seen the increasing application of micro-
fluidics, a powerful technological platform featuring small-scale
and rapid operations, to cell cultivation and subsequent analysis.
In particular, microfluidic compartmentalization has been widely
utilized. For example, microwells have been used to confine and
culture various microorganisms[26,27], including bacteria of
which the growth was quorum-sensing dependent. Micro-
fluidically generated droplets represent another strategy for
creating localized environments for diverse applications such
as cell cultivation[29,30,31,32], screening and sorting.
It should be noted that microbial communities have started
being examined using microfluidic approaches. Nevertheless,
PLoS ONE | www.plosone.org1 February 2011 | Volume 6 | Issue 2 | e17019
previous studies have focused exclusively on obtaining and
analyzing pure cultures, which did not address the key question of
how microbial interactions enable the sustenance of communities.
In this work, we aimed to make use of highly parallel micro-
droplets to co-cultivate symbiotic microbial communities. We
fabricated a microfluidic device that could readily encapsulate and
co-cultivate subsets of a community, using aqueous droplets
dispersed in a continuous oil phase. To demonstrate the
effectiveness of this approach in discovering synergistic interac-
tions among microbes, we tested it with a synthetic model system
consisting of cross-feeding E. coli mutants, which can be used to
mimic various compositions of natural microbial communities.
Encapsulation of co-cultures in microfluidic droplets
Identification of symbiotic interactions among members in a
microbial community can be facilitated by compartmentalizing
and localizing the community for co-cultivation. In this work,
microfluidic devices were fabricated for encapsulating and co-
cultivating subsets of a microbial community in monodispersed
droplets. Encapsulated microbes can grow only if the droplet
localizes symbiotic interactions in it (Fig. 1a). The device
comprised a slanted T-junction for droplet generation and a
chamber to hold droplets for cultivation (Fig. 1b,c). The slanted T-
junction is able to generate monodispersed droplets with a single
vacuum line at the outlet instead of multiple lines of pressure at the
inlets. Increasing power of the vacuum led to increase of the
frequency of droplet generation and subsequently the number of
droplets in the chamber. The achievable range of frequency was
1–500 droplets/sec. When the frequency exceeded the maximum,
the droplets were no longer mono-dispersed and co-flow of two
immiscible phases occurred. The volume of droplets was largely
determined by the channel geometry and was maintained at about
1 nl in this work.
Droplets generated from the T-junction were collected and held
in the chamber for on-chip cultivation. Surfactant was dissolved in
the oil phase and effectively stabilized the droplet surface. Up
to ,1,400 dropletscould be
10 mm65 mm chamber (Fig. 1d). After the droplets filled the
chamber, the vacuum was removed and the flow stopped
immediately. Droplets could be stably incubated for 4 days.
We hypothesized that compartmentalization of different
microbial species in a community are independent events and
for each species, the number of encapsulated cells in a droplet
follows the Poisson distribution. For experimental validation of this
hypothesis, a co-culture consisting of two differently labeled E. coli
strains, named W-and Y-, was injected into the device and the
distribution of cells was examined with fluorescence microscopy.
As shown in Fig. 2a, for each strain, the experimentally measured
distribution of droplets carrying various numbers of cells agreed
very well with calculated values using the Poisson distribution. The
average number of cells in each droplet, which corresponded to
the l parameter in the Poisson distribution, was dependent upon
the cell density in the seed culture injected into the device and the
droplet volume determined by the device configuration.
As each species’ distribution in droplets is expected to follow a
simple Poisson distribution and different species are encapsulated
independently, we could readily predict the distribution of the four
combinations (i.e. W-Y-, W-only, Y-only, and empty). Not
surprisingly, there was a very good agreement between the
calculated values and measured ones (Fig. 2b). This experimental
validation was successfully extended to the distribution of a triplet
system when a third strain, S-, was added (Fig. 2c). Therefore, the
distribution of encapsulated subsets of a microbial community is
highly predictable given the total cell concentration and
composition of the community. Accordingly, for a given
community composition, it is possible to determine the optimal
cell density of the seed culture for a fixed device to achieve a
desired droplet distribution.
packedvery tightly ina
Co-cultivation of a symbiotic pair
To mimic natural communities of interacting microbes, a
synthetic symbiotic E. coli system consisting of a tryptophan
Figure 1. A microfluidic device for microbial co-cultivation. (A) Compartmentalized co-cultures enable detection of symbiotic relations
among community members. (B) Schematic design of the microfluidic device. (C) A picture of the microfluidic device. (D) Droplets filling a large
chamber in the microfluidic device.
PLoS ONE | www.plosone.org2 February 2011 | Volume 6 | Issue 2 | e17019
auxotroph and a tyrosine auxotroph was constructed. Each
auxotroph is unable to synthesize the corresponding amino acid
and hence cannot survive in minimal media. However, when both
auxotrophs are present, they can grow in the minimal medium by
cross-feeding (Fig. 3a). To monitor the co-culture composition,
each strain was labeled with a fluorescent protein and counted by
On-chip co-cultivation of the tryptophan auxotroph (abbrevi-
ated to W-) and the tyrosine auxotroph (abbreviated to Y-)
demonstrated that droplets could effectively compartmentalize co-
cultures of microbes. Seed cultures of W-and Y-were diluted with
the minimal medium and mixed properly such that the W-:Y-ratio
was 1:1 and upon injection into the device, the average cell
number per droplet was about 0.6. A total of 608 droplets were
generated in this experiment. Of those, 317 were empty. 83 and
150 droplets had W-only and Y-only, respectively. 58 consisted of
the W-and Y-pair.
After 18 hours of cultivation, only the cells in the droplets
carrying both W-and Y-cells were growing well (Fig. 3b,c). We
noted that some of the droplets carrying W-or Y-only were
adjacent to droplets carrying the W-and Y-pair during
cultivation, but cells in these droplets did not grow. This implied
that the diffusion of amino acids did not occur across the droplet
boundaries. In other words, the oil-water interface effectively
blocked molecular diffusion between different droplets and
therefore the droplets could generate highly parallelized and
localized co-cultivation environments for detecting symbiotic
relationships in a large and complex microbial community.
On-chip droplet-based co-cultivation could further distinguish
stronger symbiotic relationships from weaker ones among subsets
of microbes. This could be demonstrated by examining two
different strains of the tryptophan auxotroph when they were
paired with the tyrosine auxotroph. We made use of a K-12 W-
strain and an EcNR W-strain. Both grew with E. coli K-12 Y-in
the minimal medium, while the K-12 W-and Y-pair co-grew 50%
faster than the EcNR W-and Y-pair in macro-scale tube cultures
(growth rates: 0.18960.011 hr21versus 0.12660.004 hr21). As
shown in Fig. 4, when we injected a mixed culture of K-12 W-,
EcNR W-, and Y-with a ratio of 1:1:10 into the device, cells in
droplets containing K-12 W-and Y-(Fig. 4a) grew significantly
better than those in droplets containing EcNR W-and Y-(Fig. 4b).
Co-cultivation of a triplet system
To mimic the complexity of natural communities more
realistically, we introduced a third amino acid auxotroph, a serine
auxotroph, into the system (Fig. 5a). The serine auxotroph
(abbreviated to S-) was previously found to form no cross-feeding
relationship with W-or Y-and hence could be considered as the
background or noise in the community. We first examined the
simplest case where the ratio of S-: W-: Y-was 1:1:1. The average
number of cells in each droplet was controlled to be three. For
counting of cells and monitoring of growth, S-, W-and Y-were
labeled with CFP, mCherry and EYFP, respectively. In this triplet
system, eight different combinations were possible. A total of 816
droplets were generated and the number of droplets carrying each
combination is shown in Table 1. As expected, among these
droplets, only the cells in the droplets carrying the W-and Y-pair
or the S-, W-and Y-triplet were able to grow, as shown in Table 1
and Fig. 5b. In particular, 112 droplets carried W-and Y-initially
and 109 of them (97%) supported growth very well after 18 hours.
This result showed that the droplet device was able to detect
symbiotic relationships among subsets of members despite the
presence of other microbes in the community.
Figure 2. Comparisons between experiments and calculations
for cell distribution in droplets. Calculations were based on the
Poisson distribution. (A) Numbers of droplets carrying different
numbers of cells. (B) Numbers of droplets carrying four different
combinations of a two-strain system. (C) Numbers of droplets carrying
eight different combinations of a three-strain system.
PLoS ONE | www.plosone.org3February 2011 | Volume 6 | Issue 2 | e17019
In natural microbial communities, symbiotic partners might
account for a small fraction of the total population. To mimic such
conditions in nature, we further examined two other compositions
of the E. coli triplet system. In one scenario, one partner of the
symbiotic pair is a rare species; in the other, both partners are rare.
In both cases, we tested the limit to which the device was able to
detect the symbiosis between W-and Y-by generating droplets
containing both and hence supporting co-growth of the pair. For
the first case, we examined a mixed culture with the ratio of S-: W-:
Y-=50:50:1, in which Y-is a rare species accounting for about 1%
of the total population. Based on simulation of cell distribution in
droplets, we selected the average number of cells in each droplet to
be five. To generate this average cell number per droplet, we
estimated the density of the mixed culture under microscope using
a Petroff-Hausser counting chamber and then diluted accordingly
before injecting it into the device. Using a device with a
3 mm610 mm incubation chamber, we generated a total of 880
droplets. As shown in Table 1b, because of the dominance of S-
and W-in the population, the majority of the droplets contain
either one or both of them, without the presence of the rare Y-.
Nevertheless, four droplets turned out to contain the W-/Y-pair,
eighteen droplets encapsulated all three strains, and most of them
(19 out of 22) showed well-sustained co-growth. It should be noted
that for our synthetic system, the W-/Y-pair can co-grow when S-
is present and hence the above two types of droplets (i.e. W-/Y-
and S-/W-/Y-) can both detect the existing symbiotic relationship.
However, in natural microbial communities, due to negative
interactions with other species, symbiotic partners may not be
able to grow when other species are present. In this case, only the
droplets that contain the symbiotic partners (e.g. W-/Y- in our
model system) are desirable for supporting co-cultivation and
detection of symbiotic relationships.
In the second case, we focused on the ratio of S-: W-:
Y-=30:1:1, which exemplified symbiotic relationships existing
between two rare species (here, each partner is about 3% of the
total population). We chose to encapsulate an average of three cells
in each droplet and generated a total of 977 droplets on a device
with a 3 mm610 mm chamber. In this scenario, a large fraction of
the droplets contain S-, as detailed in Table 1c. Nevertheless, we
managed to observe six droplets that encapsulated the W-/Y-pair,
including one containing only these two strains. All of these
droplets supported the co-growth of W-/Y-very well. The above
results demonstrated that our device and co-cultivation method
can effectively capture and amplify rare species in a microbial
community and detect their symbiotic relationships with either
abundant or other rare species.
In this work, we have demonstrated that microfluidically
generated droplets can be effectively utilized to co-cultivate
microbes and detect symbiotic relationships. Two features of our
microfluidic device contributed to this effectiveness. First, due to its
small dimensions and rapid operation, our droplets based device
can achieve compartmentalization of microbial communities in a
highly parallel and automated manner. Second, the interface
between the aqueous phase in the droplet and the oil phase
prevents molecular exchanges, and hence the cultivation environ-
ment in individual droplets is completely localized.
We used a synthetic E. coli symbiotic co-culture as a model
system in this proof-of-concept study. Nevertheless, the platform
we presented here for on-chip co-cultivation can be readily applied
to a wide range of natural microbial communities. As shown in this
work, the distribution of cells in droplets is highly predictable.
Figure 3. On-chip cultivation of a cross-feeding pair. (A) A synthetic symbiotic system consisting of two cross-feeding amino acid auxotrophs.
(B) A section of the large cultivation chamber illustrating a number of droplets carrying four combinations of the two-strain system. E. coli strain Y-is
labeled with yellow fluorescence (EYFP) and W-with red fluorescence (mCherry). Left – before cultivation, Right – Pictures after 18-hour cultivation.
(C) Comparison of four individual droplets from Panel (B).
PLoS ONE | www.plosone.org4 February 2011 | Volume 6 | Issue 2 | e17019
Therefore, for a given microbial community with certain total
density and composition, the design (e.g. droplet and chamber
size) and operation (e.g. dilution ratio) of our microfluidic device
can be optimized to co-cultivate and examine community
members at different levels of abundance. For example, in this
work, with up to 1,000 droplets, we could adjust the dilution ratio
to detect the synthetic symbiotic interaction when one or both of
the pair was very rare in the population. In addition, we showed
that different extent of symbiosis could also be distinguished. Thus,
our work suggests a promising new approach for cultivating
microbes and for understanding microbial interactions. We can
use this approach to cultivate and isolate various microbes of
which the growth requires support (e.g. through signaling) from
other species in the community. The cell cultures can then be
further studied using a variety of characterization and analysis
methods such as (meta)genome sequencing. Moreover, the co-
growth data obtained from this co-cultivation approach will reveal
Figure 4. Comparison of a fast growing pair (K-12 W-and Y-)
and a slow growing pair (EcNR1 W-and Y-) on the same device.
(A) Three droplets carrying the pair of E. coli K-12 W-expressing
mCherry and Y-(not labeled with fluorescence). Top panels – before
cultivation. Bottom panels – after 18-hour cultivation. (B) Three droplets
carrying the pair of E. coli EcNR1 W-expressing GFP and K-12 Y-. Top
panels – before cultivation. Bottom panels – after 18-hour cultivation.
Figure 5. On-chip cultivation of a triplet system. (A) A synthetic system of three amino acid auxotrophs (W-, Y-, and S-). Only W-and Y-forms a
symbiotic relationship. (B) Eight droplets illustrating all the combinations of the triplet system. Top panels are pictures taken before the cultivation
and bottom panels are at 18 hours.
Table 1. Number of droplets carrying various subsets of the
no growth436273 8010612733
no growth 40 1531382 515612
no growth 3279821 787000
(a) S-: W-: Y-=1:1:1 (b) S-: W-: Y-=50:50:1 (c) S-: W-: Y-=30:1:1.
PLoS ONE | www.plosone.org5 February 2011 | Volume 6 | Issue 2 | e17019
positive relationships among community members, such as those
between Bifidobacterium adolescentis and butyrate-producing anaer-
obes in the human gut, and negative ones. Elucidation of these
microbes and their interactions might have important implications
for many applications such as diagnostics and treatment of
This work represents an initial step towards the elucidation of
microbe-microbe-environment interactions of complex communi-
ties based on microdroplet co-cultivation and characterization. To
fulfill this long-term goal and to apply this approach to real
microbial communities, we have identified two key tasks that
require further efforts. First, automated droplet sorting and
retrieval will enable us to distinguish droplets with well-developed
mixed cultures from those with little growth and to obtain each of
them individually. This can be achieved by coupling microscopy
with flow control of droplets. Second, when studying natural
microbial communities, in which none of the cells is labelled, we
need to characterize the retrieved droplet-mediated mixed
cultures. Three genetic approaches with varying levels of details
are suitable for this purpose. Terminal restriction fragment length
polymorphism (T-RFLP) of 16 s rDNA is a simple method that
can rapidly determine how many species are present in the
cultivated communities. Sequencing of 16 s rRNA will identify
the cultured microbes at a species level. Finally, whole genome
amplification followed by metagenome sequencing will potentially
lead to comprehensive and detailed understanding of the genetic
basis underlying the microbial interactions[39,40]. We are
currently investigating these microfluidic developments and off-
chip characterization methods, which will facilitate the scale-up of
the approach presented in this work for application to complex
natural microbial communities.
Materials and Methods
We used glass devices. Channels were fabricated using general
photolithography processes. A glass wafer was prepared with Cr-
Au deposition and AZ1518 spin coating. The pattern of the Cr
mask was transferred to the AZ1518-coated glass wafer with the
UV exposure of LI 300 for 30 seconds. After developing for 1
minute in MF319 developer, Au and Cr were etched for 2
minutes. The glass wafer was wet-etched by HF, until the depth of
the channel reach 50 mm. The channel depth was measured
periodically by depth profiler during the etching process. After
dicing the individual devices, holes for inlets and outlets were
electrochemically drilled in sodium hydroxide solution. To make
the surface hydrophobic, a 2 mm-thick Parylene film was deposited
on the channel and a cover slip. Afterwards, the channel and the
cover slip were bonded with UV glue. Glass tubes were attached
with UV glue at the holes as reservoirs of inlets and outlets, and
syringe tips were fixed to the oil inlet and outlet reservoirs with
epoxy to connect the device to vacuum source and oil reservoirs.
Before using the devices for the cultivation, all devices were
exposed to UV for at least one hour for sterilization. All devices
were regenerated after each use. They were heated in a 540uC
furnace for 2 hours, followed by cleaning in Piranha solution of
H2O2:H2SO4=1:2. Parylene coating, bonding with UV glue,
attaching reservoirs and sterilization were repeated as described
Construction of fluorescently-labeled synthetic symbiotic
Four E. coli strains were used in this work and all were amino
acid auxotrophs constructed through the deletion of genes or
operons encoding key enzymes in the amino acid biosynthesis
pathways. K-12 W-, K-12 Y-, and K-12 S-were constructed by
deleting trpE, tyrA, and serA, respectively, in E. coli K-12. In
addition, tyrR was deleted in K-12 W-to increase the co-culture
fitness when it is paired with K-12 Y-. All of the above gene
deletions were carried out via P1 transduction with single-gene
knock-out E. coli mutants from the Keio collection. After the
transduction, the selection marker (kan gene) was deleted by
transformation with the plasmid PCP20. The EcNR W-strain was
previously constructed by recombinogenic substitution of the
trpLEDCBA operon with a selection marker (cat gene) in a
derivative strain of E. coli MG1655 harboring a l Red prophage.
This tryptophan auxotroph strain grew at a slower rate when
paired with K-12 Y-.
For labeling of different strains, four fluorescent proteins,
mCherry, EYFP, CFP, and GFP, were utilized. mCherry and
GFP were inserted into pET24b and EYFP into pET17b
plasmids, respectively. The PBADpromoter was inserted in front
of the fluorescent gene, deleting the original T7 promoter and lac
operator. Resulted plasmids were transformed into different
strains as needed. CFP was integrated into the chromosome at
the galK locus via P1 transduction with RP22 as the donor
To prepare seed cultures for on-chip cultivation, cells expressing
mCherry, GFP and EYFP were cultivated overnight in LB media
containing 0.4% arabinose and cells labeled with CFP in LB
media containing 0.2 mM IPTG. Then each strain was harvested
and diluted 100 times in M9 minimal medium containing 0.4%
arabinose and 0.2 mM IPTG. After the cell density of each seed
culture was estimated using a Petroff-Hausser counting chamber
and a fluorescence microscope, the diluted seed cultures were
mixed to obtain the desired ratios.
Encapsulation of microbes
A PFPE-PEG block copolymer surfactant (RainDance
Technologies) was dissolved in fluorinated oil HFE-7500 (3 M)
at a final concentration of 2% wt/wt. The oil phase was supplied
in a syringe connected to the device. After the device was
connected to the vacuum source, 30 ml of diluted cell mixture was
added into the aqueous-phase inlet reservoir. Using LabView
interface, continuous vacuum was turned on, and the power of the
vacuum was increased gradually to 150–300 mmHg. After enough
droplets were generated, vacuum was turned off. All syringes and
connections were removed from the device. 5 ml of mineral oil was
added to each reservoir to prevent the evaporation of fluorinated
Cultivation and monitoring of microbes
As soon as droplet generation was completed, the device was
examined by microscopy. Pictures at the beginning and the end of
cultivation were taken by Olympus BX-51 and DP-71 with 20x
objective lens. Exposure time and ISO were 0.25 sec/800,
0.8 sec/1600, and 0.2 sec/400 for mCherry, EYFP and CFP,
respectively. For all pictures of CFP-expressing cells, autolevel and
autocolor functions in Adobe Photoshop were applied to enhance
the contrast. ImageJ was used to combine pictures from different
channels. The device was placed in an incubator of 37uC for
cultivation and pictures were taken as needed.
We thank RainDance Technologies for providing us with the PFPE-PEG
block copolymer surfactant and Michael B. Elowitz for sharing with us the
RP22 strain. We are grateful to Michael J. Solomon and Chien-Ching
PLoS ONE | www.plosone.org6February 2011 | Volume 6 | Issue 2 | e17019
Lilian Hsiao at the University of Michigan for their help in combining
images from multiple channels with ImageJ. We also thank Vincent B.
Young and Betsy Foxman for feedbacks on this manuscript.
Conceived and designed the experiments: JP MAB XNL. Performed the
experiments: JP. Analyzed the data: JP MAB XNL. Contributed reagents/
materials/analysis tools: AK. Wrote the paper: JP MAB XNL.
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PLoS ONE | www.plosone.org7 February 2011 | Volume 6 | Issue 2 | e17019