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Micropatterning bacterial suspensions using aqueous two phase systems† Toshiyuki Yaguchi,a Siseon Lee,a Woon Sun Choi,b Dasol Kim,a Taesung Kim,*b Robert J. Mitchell*a and Shuichi Takayama*ac
Downloaded by University of Michigan Library on 23 May 2011 Published on 13 September 2010 on http://pubs.rsc.org | doi:10.1039/C0AN00464B
Received 1st July 2010, Accepted 7th August 2010 DOI: 10.1039/c0an00464b Using an aqueous two-phase system comprised of dextran and polyethylene glycol, this article describes the stable spatial patterning of sub-microlitre droplets of bacterial suspensions. Microdroplets of different types of bacterial populations are positioned and maintained adjacent to each other without significant dispersion even though the bacteria are in suspension and not surface bound. Small molecules, in contrast, diffuse relatively freely between the two aqueous phases. The usefulness of these capabilities is demonstrated by generating a small array of suspensions containing different Escherichia coli strains engineered to respond fluorescently or luminescently to different chemical stimuli. When a chemical stimulus is presented, this droplet array produces a pattern of bacterial ‘‘illumination’’ that reflects the type of chemical to which the array was exposed.
1. Introduction Physically segregated but chemically connected patterns of different bacterial populations are useful in generating bacteriabased biosensors1–3 as well as in studying the functions of bacterial and other cellular communities.4–6 Typically, such bacterial arrays or communities are generated by immobilizing the bacteria onto a surface by adhesion,7,8 or by segregation of the bacterial micro-colonies using filter membranes5 or semisolid matrices such as hydrogels.2,6 Although useful, surface immobilized bacteria can detach, outgrow their patterns, or be unnaturally limited in growth and mobility.8 Use of filter membranes or hydrogel microstructures requires micro-fabrication procedures and can limit optical access because of the filter or other structures. It would be useful, therefore, to be able to generate chemically inter-connected micro-colonies of different types of bacterial suspensions without the use of micro-fabrication, filters, hydrogel structures, or surface immobilization. Bacteria in suspension, however, will typically disperse due to diffusion, convection, as well as due to the motility of the bacteria themselves, thus, precluding spatial confinement. Here, we demonstrate that aqueous two-phase systems (ATPSs) can be used to stably localize even very high concentrations of Escherichia coli bacteria as distinct spatially confined micro-colonies that are segregated from each other but chemically communicating. We further demonstrate the usefulness of this technology for chemical detection by generating a small array of bacterial suspensions a School of Nano-biotechnology and Chemical Engineering, Ulsan National Institute of Science and Technology, Banyeon-ri 100, Ulsan, 689-798, Republic of Korea. E-mail:
[email protected]; Fax: +82-52-217-2509; Tel: +82-52-217-2509 b School of Mechanical and Advanced Materials Engineering, Ulsan National Institute of Science and Technology, Banyeon-ri 100, Ulsan, 689-798, Republic of Korea. E-mail:
[email protected]; Fax: +82-52217-2309; Tel: +82-52-217-2313 c Dept Biomedical Engineering and Macromolecular Science & Engineering Program, University of Michigan, 2200 Bonisteel Blvd, Ann Arbor, MI, 48109-2099, USA. E-mail:
[email protected]; Fax: +1-734-9361905; Tel: +1-734-615-5539 † Electronic supplementary information (ESI) available: 3D droplet video taken by confocal microscopy. See DOI: 10.1039/c0an00464b
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that respond to different chemicals through changes in their fluorescent or luminescent output.
2. Experimental 2.1 Preparation of ATPS solutions ATPS solutions were prepared by mixing 7.5% polyethylene glycol (PEG, Mw: 8000, Sigma-Aldrich, Co.) and 7.5% dextran (DEX T20, Mw: 20 000, Pharmacosmos) in Luria Broth (LB) medium, then allowing the two-phases that result to separate by gravity. This ATPS formulation utilizes relatively low molecular weight DEX and PEG which reduces the viscosity of the resulting DEX-rich and PEG-rich phases of the resulting ATPS solutions. For ATPS patterning by manual pipetting, the lower viscosity is advantageous because the dispensing occurs quickly and accommodates a ‘‘less steady’’ hand during the patterning process. 2.2 E. coli transformation and culture We prepared 6 types of bacteria. Two types of bacteria that alter expression of green fluorescent protein (GFP) in response to acyl homoserine lactone (AHL) were prepared. Competent cells, strain DH5a, were prepared and transformed with plasmids t9002 (receiver) and k137019 (inverse-receiver) transferred from the Registry of Standard Biological Parts (www.partsregistry.org), respectively. The ‘‘receiver’’ cell is activated to express GFP by AHL, while the ‘‘inverse-receiver’’ cell that initially expresses GFP constitutively is inhibited, suppressing additional GFP expression.6 Two types of bacteria that constitutively express fluorescent proteins were prepared. E. coli K12 MG1655 were transformed with plasmids pAmCyan (Clontech, USA), which confers ampicillin resistance, or pHKT3, which confers tetracycline resistance.9 These strains express the cyan fluorescence protein (CFP) and red fluorescence protein (RFP), respectively. Two types of E. coli K12 MG1655 bioluminescent strains were also prepared. For this, plasmid pUCD61510 was digested with KpnI and self-ligated without the 7 kb fragment.11 This plasmid, This journal is ª The Royal Society of Chemistry 2010
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referred, to as pUCDK, was transformed into E. coli MG1655, giving a highly bioluminescent strain, MKan3. This strain was used for both an analysis of the PEG and DEX toxicity and within the bacterial array sensing experiments. The second bioluminescent strain, strain Rec3 (inducible), had the recA promoter cloned upstream of the luxCDABE operon in pUCD615. This was done using the primers: recA-BF, 50 -ACTTAAGGATCCAGAGAAGCCTGTCGGCAC-30 , and recAER, 50 -AGCTTTGAATTCCGCTTTCTGTTTGTTTT-30 . After PCR amplifying the promoter region, the PCR product was digested using BamHI and EcoRI and ligated into pUCD615, which was digested using the same enzymes. The ‘‘inducible’’ strain increases luminescence in response to DNA damage. For the growth of these strains, each E. coli culture was grown overnight on Luria agar plates with the appropriate antibiotic at 30 C. A single colony was used to inoculate 5 mL LB broth with either 50 mg mL1 amplicillin or 5 mg mL1 tetracycline added. The cultures were then grown overnight (16 h) with vigorous aeration (200 rmp in a rotary shaker), resulting in OD600 ¼ 1.5–2. A portion of the culture, 4.0 mL, was centrifuged at 3000 rcf for 5 min and the pellet was resuspended with the DEX-rich phase to give bacterial suspensions with the desired cell densities. 2.3 Preparation of bacterial suspensions in ATPS solutions Bacterial suspensions were prepared by centrifugation of the bacterial cultures at 3000 rcf, removal of the supernatant, and resuspension of the bacterial cells in the PEG or DEX phase solutions. The removal of the culture media was important to allow preparation of bacterial suspensions with very well defined ATPS solution compositions and also to allow, when necessary, the preparation of bacterial suspensions with a higher density compared to the original culture. 2.4 Evaluation of bacterial growth and viability in the PEG and DEX phases The toxicity and viability tests were performed using overnight cultures of a highly bioluminescent E. coli, strain MKan3, grown in LB media with ampicillin. The cultures were mixed 1 : 10 with aqueous solutions of PEG 8K, DEX T20 or the PEG or DEXrich phases of ATPSs prepared by mixing and separating the two polymers. Aliquots (200 mL) of the mixture were transferred to the wells of a white 96-well plate (Greiner, USA) and analyzed using the GloMax Multi+ Plate Reader (Promega), which was set to read the bioluminescence from the samples every hour for 4 hours. The temperature of the Glomax was set to 30 C and the plate was set to shake (medium, 30 s) before each reading. The bioluminescence data were collected and analyzed using Sigma Plot v.11. 2.5 Bacterial micropatterning in ATPS Small (volume range: 0.1–2.5 mL) droplets of bacteria, suspended in the DEX phase, were patterned in PEG phases using a conventional manual pipettor (Eppendorf) to dispense the DEX phase at desired positions. The ability to stably maintain the localization of bacterial microdroplets was analyzed by patterning 0.2 mL suspensions of the CFP-expressing strain at This journal is ª The Royal Society of Chemistry 2010
different cell densities (1 106 to 16 106 cells per mL) and imaging the bacteria positions periodically using fluorescence imaging. DEX phase bacterial suspension droplets in PEG phase was also imaged using a Carl Zeiss LSM700 confocal microscope operated by a ZEN 2009 software to visualize the 3D distribution of bacteria in the droplet and demonstrate clear partitioning of bacteria within the DEX phase. 2.6 Evaluation of rhodamine B diffusion in ATPS Small (volume: 0.2 mL) droplets of rhodamine B (0.01 mM) dissolved in the DEX phase were dispensed into the PEG phase using a pipettor. The diffusion of rhodamine B was visualized fluorescently. 2.7 Bacteria microdroplet array for biochemical sensing Five different genetically engineered E. coli were arrayed. These include bacteria that: (i) responds to AHL by increasing expression of GFP (we refer to these bacteria as the ‘‘receiver’’), (ii) respond to AHL by decreasing their expression of GFP (inverse-receiver), (iii) constitutively expresses bioluminescence (luminescent), (iv) responds to mutagens through a higher bioluminescence (inducible) and (v) constitutively expresses CFP (cyan). Suspensions of each bacteria were prepared in the DEX phase as described above (Section 2.3) and 0.2 mL droplets of each suspension were arrayed manually, adjacent to each other, into PEG phases containing a mutagen (mitomycin C), AHL, or neither chemical. Changes in fluorescence and bioluminescence were measured using the magnification 0.7 setting using an Olympus SZX16 stereoscope, a DP72 CCD camera operated by the DP2-BSW software over the course of 4 hours. Experiments were performed at least in triplicate and results are expressed as relative change in luminescence or fluorescence between time 0 and 4 h. Statistical analysis was performed by ANOVA followed by Tukey tests. Statistically significantly different groups are designated with different letters (a, b, c, and d) in the bar graphs in Fig. 4.
3. Results and discussion ATPSs have been used for decades to perform gentle bioseparation of molecules, organelles, and cells.12–16 In applications with bacteria, ATPSs have been used not only for bioseparations but also for continuous extraction combined with bacterial cultures.17 These systems, however, target macroscale cultures and the manipulation of bacteria. The use of ATPS for separations in microchannels has been reported18–20 but not for stable micropatterning. Recently, we described the use of nanolitre scale ATPS droplets to pattern surface adherent mammalian cells21 and also to pattern polymeric and viral nucleic acid reagents.22 In this article we describe an adaptation of this ATPS micropatterning technology to pattern micro-colonies of bacterial suspensions. 3.1 General procedure for bacterial suspension patterning The general procedure is illustrated in Fig. 1. Pipettes containing bacteria suspended in the DEX-rich phase are gently inserted into the PEG-rich phase. After positioning the pipette tips at the Analyst, 2010, 135, 2848–2852 | 2849
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3.2 Confirming non-toxicity of ATPS solutions For biological studies and functional applications of the micropatterned bacterial suspensions, it is critical to confirm the viability of the bacteria in these aqueous two-phase formulations. For this reason, we performed toxicity analysis using a highly bioluminescent strain of E. coli. A bioluminescent strain was selected since the bioluminescence (BL) is integrally tied to the health of the cell23,24 and can be used to evaluate the toxicity of an aqueous sample.25 The results, shown in Fig. 2, indicate that the PEG solutions lead to the greatest BL inhibition, with an approximately 40% lower BL value than the control after 1 hour. In comparison, the DEX solutions were slightly better, only causing a 30% loss. Similar losses were seen from the ATPSprepared samples (Fig. 2). The loss in bioluminescence was only temporary, however, since the BL values from later time points are very similar for all the samples. 3.3 Stable localization of bacterial suspensions but with free diffusion of small molecules
Fig. 1 Procedure for micropatterning bacterial suspensions using aqueous two-phase systems (ATPSs) comprised of dextran (DEX) and polyethylene glycol (PEG). (A) Pipettes containing suspensions of different bacteria (cyan or red fluorescent) suspended in the DEX-rich phase is positioned inside a culture dish filled with the PEG-rich phase. At the desired positions, the suspensions are dispensed from the pipettes to produce droplets of the two different types of bacterial suspensions which are physically segregated. (B) Fluorescent micrograph of actual bacterial suspensions patterned using this technique. Bar ¼ 500 mm. (C) 3D droplet image taken by confocal microscopy. The 3D droplet image clearly shows that the bacteria are all inside the curved outline of the DEX phase droplet. Bar ¼ 100 mm. (D) An example with 16 CFP-expressing bacterial suspension droplets patterned adjacent to each other. Bar ¼ 1 mm.
desired positions, the bacterial suspensions are simply dispensed. Because of the partitioning preference of the bacteria for the DEX-rich phase, the bacteria stay localized within the DEX droplet without dispersion (Fig. 1C), even though the bacteria are in suspension and are not surface immobilized. Suspensions of different genetically modified E. coli can be positioned adjacent to each other with no mixing between the two suspensions as shown in Fig. 1B. It is also possible to pattern multiple droplets such as shown in Fig. 1D where 16 droplets of bacterial suspensions were produced. Dispensing at the lowest possible volumes using manual pipettors does produce some drop to drop variability. The use of more accurate dispensing tools should allow for higher reproducibility for applications that require strict quantitative control. 2850 | Analyst, 2010, 135, 2848–2852
The density and total number of bacteria regulate a variety of important functions, such as quorum sensing for biofilm formation.26–29 Additionally, bacteria proliferate rapidly. It is thus important that the droplets of bacterial suspension maintain their micro-colony patterns over a wide range of bacteria densities. Fig. 3 shows a comparison of bacterial suspension micro-colonies patterned over a range of relatively high concentrations. In sharp contrast to the rapid dispersion of the bacterial micro-colony when the bacterial suspension in DEX is simply dispensed into another DEX phase (Fig. 3A), 0.2 mL bacterial suspensions of different densities up to 16 109 cells per mL take on and stably maintain a rounded shape when the DEX suspension is dispensed into the PEG phase (Fig. 3C). Importantly, even for the very high bacteria densities, the aqueous two-phase patterned micro-colonies suspended in DEX
Fig. 2 Use of bioluminescence to evaluate the effects of DEX 20 000 and PEG 8000 and prepared ATPS solutions of these two compounds on the bacterial health. The results are shown as the normalized bioluminescence based upon the initial reading (BL0 at t ¼ 0 hours). Control: LB media. DEX 20: LB media with 20% w/w DEX T20. PEG 8: LB media with 20% w/w PEG 8000. ATPS D20: DEX-rich phase of 7.5% DEX T20 and 7.5% PEG 8000 ATPS. ATPS P8: the PEG-rich phase from the same ATPS. Error bars are the standard deviations calculated from triplicate samples.
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Fig. 3 Evaluating the stable localization of E. coli suspensions in ATPSs and the contrast with the rapid diffusion of small molecules in ATPS or bacteria in just the DEX phase. (A) E. coli suspensions will disperse out rapidly when a DEX suspension of the bacteria is simply dispensed into a DEX phase. (B) Small molecules such as rhodamine B freely diffuse and are not selectively partitioned even in ATPSs. (C) In contrast, E. coli suspensions in DEX when dispensed into PEG remain localized even at high cell concentrations. Bar ¼ 1 mm.
droplets maintained a stable spatial localization within the PEG phase. Although there are slight differences between the exact pattern of bacterial fluorescence at time 0 and time 60 minutes, this can be attributed to the slight movement of the droplets during transport of the plates back and forth between the stereoscope used for imaging and the bacteria incubator. It is interesting to contrast this ability of the PEG–DEX ATPS to localize bacteria with the relatively free diffusion of small molecules. Rhodamine B, for example, diffuses very rapidly from DEX phase droplets into the PEG phase (Fig. 3B). This is consistent with previous observations where small molecules are typically not selectively partitioned in ATPSs.18 3.4 Bacterial micro-colony biosensor array Utilizing the ability of ATPSs to spatially localize bacteria in an environment where there is free diffusion and relatively little selective partitioning of small molecules, we demonstrated the preparation of bacterial suspension arrays for biochemical detection. We dispensed, into a PEG phase, five 0.2 mL DEX droplets adjacent to each other where each droplet contained suspensions of different bacterial strains (Fig. 4A). Each bacteria micro-colony becomes a biosensor droplet where, depending on the type of bacteria suspended, the droplet are expected to (a) constitutively expresses bioluminescence, (b) constitutively expresses CFP, (c) increase expression of GFP in response to AHL (receiver), (d) decrease expression of GFP in response to AHL (inverse-receiver), and (e) increase This journal is ª The Royal Society of Chemistry 2010
Fig. 4 A bacterial suspension-based biosensor array. (A) Schematic description and an optical micrograph of the 200 nanolitre bacterial suspensions patterned using ATPS technology. (a) E. coli constitutively expressing bioluminescence also referred to as ‘‘Lumi’’. (b) Cells constitutively expressing cyan fluorescent protein (cyan). (c) Cells that increase GFP expression in response to AHL (Rec). (d) Cells that decrease GFP expression in response to AHL (Inv-Rec). (e) Cells that increase bioluminescence in response to mutagens (Ind-Lumi). Bar ¼ 1 mm. (B) Bar graph showing normalized changes in intensity of luminescence or fluorescence of the bacterial suspension droplets comparing readings at time 0 and 4 h with no chemical stimulation. Inset shows luminescence (upper) and fluorescence (lower) images of the array at 4 h. (C) Normalized changes in intensity of luminescence or fluorescence with mitomycin C stimulation. Inset shows micrographs of the luminescence observed for the ‘‘Ind-Lumi’’ bacteria droplet at time 0 and 4 h (a and c ¼ P < 0.05). (D) Normalized changes in intensity of luminescence or fluorescence with AHL stimulation. Inset shows micrographs of the fluorescence observed for the ‘‘Rec’’ bacteria droplet at time 0 and 4 h (a, b, c, and d ¼ P < 0.05).
bioluminescence in response to mutagens (inducible). Fig. 4B shows the micrographs and bar graphs of the behaviour of the bacterial biosensor array with no chemical stimulation. With no chemical added, we found that there was no significant change in fluorescence or luminescence between time 0 and 4 h. Fig. 4C shows micrographs of the response of the bacterial biosensor array over a 4 h period when the PEG phase is spiked with the Analyst, 2010, 135, 2848–2852 | 2851
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mutagen mitomycin C. The notable readout here is the increase in the bioluminescence of the ‘‘inducible’’ bacteria. The BL from the constitutively bioluminescent bacteria decreases, which is consistent with a toxic response brought on by the mutagen. Fig. 4D shows the response of the bacterial biosensor array over a 4 h period when the PEG phase is spiked with AHL. The notable readout here is the increase in fluorescence of the ‘‘receiver’’. We also found, unexpectedly, a general trend for decreased fluorescence and luminescence in the presence of AHL. Although E. coli does not possess the gene necessary to produce AHLs, it does express a protein (SdiA) with an amino acid sequence similar to that of the LuxR-type transcriptional activators,30 which are involved in sensing for AHLs. Furthermore, recent work shows that SdiA does indeed mediate AHL-triggered gene expression changes in E. coli,31,32 providing a plausible explanation for our ‘‘unexpected’’ observations. These results demonstrate the usefulness of ATPS micropatterning technology to generate bacterial micro-colony arrays for biochemical sensing. The ability to conveniently perform multiplexed tests using small amounts of reagents and cells in suspension also opens new possibilities for discovery-driven research where unexpected or unpredictable insights may be obtained. The ability for bacterial suspensions in these arrays to respond to AHLs also suggests potential utility of the ATPSbased bacterial micropatterning technology for engineering bacterial communities with spatially segregated but chemically communicating bacteria micro-colony networks.5
4. Conclusions We describe a method to generate stable microarrays of different bacterial suspensions in ATPSs. The ability to stably micropattern freely floating bacteria in fully aqueous solutions is somewhat counter-intuitive and intriguing. Each bacterial suspension is stably confined within their DEX-rich microdroplets by selective partitioning, yet they are chemically connected to each other through the PEG-rich phase, where small molecules can freely diffuse. By arraying a number of genetically engineered biosensor bacteria, a bacteria-based biochemical sensor was developed that responds to different chemicals by altering their illumination properties. The advantages of the ATPS bacterial micropatterning methods include the simplicity of the procedures, the high optical accessibility afforded by the absence of filters or microstructures often required in other methods for segregating bacterial micro-colonies from each other, and the ability to use normal bacteria culture media, such as M9 or LB media, by simply adding PEG and DEX. There is no micro-fabrication or special tools that are required, just some commercially available polymers. Thus, the technique should be easy to implement by all. There are some limitations that one does need to consider, such as the movement of droplets that can result if one jolts or tilts the culture dish significantly and the differences in the partitioning behavior of different bacterial species.18 These limitations, however, should generally be overcome through careful handling and, if needed, through adjusting the types and concentrations of the polymers used to make the ATPSs.33 We believe the technology will be broadly useful for biologists and technologists for studying bacterial community interactions as well as for making bacteria-based biosensors. 2852 | Analyst, 2010, 135, 2848–2852
Acknowledgements This work was supported by WCU (World Class University) program funded by the Ministry of Education, Science and Technology (R322008000200540).
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