Nanoscale

PAPER

Cite this: Nanoscale, 2017, 9, 3485

Received 20th October 2016,Accepted 6th February 2017

DOI: 10.1039/c6nr08224f

rsc.li/nanoscale

High-throughput screening of microchip-synthesized genes in programmable double-emulsion droplets†

H. F. Chan,‡§a,b S. Ma,§a,c J. Tiana,c,d and K. W. Leong*a,b,e

The rapid advances in synthetic biology and biotechnology are increasingly demanding high-through-

put screening technology, such as screening of the functionalities of synthetic genes for optimization

of protein expression. Compartmentalization of single cells in water-in-oil (W/O) emulsion droplets

allows screening of a vast number of individualized assays, and recent advances in automated

microfluidic devices further help realize the potential of droplet technology for high-throughput

screening. However these single-emulsion droplets are incompatible with aqueous phase analysis and

the inner droplet environment cannot easily communicate with the external phase. We present a high-

throughput, miniaturized screening platform for microchip-synthesized genes using microfluidics-

generated water-in-oil-in-water (W/O/W) double emulsion (DE) droplets that overcome these limit-

ations. Synthetic gene variants of fluorescent proteins are synthesized with a custom-built microarray

inkjet synthesizer, which are then screened for expression in Escherichia coli (E. coli) cells. Bacteria

bearing individual fluorescent gene variants are encapsulated as single cells into DE droplets

where fluorescence signals are enhanced by 100 times within 24 h of proliferation. Enrichment of

functionally-correct genes by employing an error correction method is demonstrated by screening DE

droplets containing fluorescent clones of bacteria with the red fluorescent protein (rfp) gene.

Permeation of isopropyl β-D-1-thiogalactopyranoside (IPTG) through the thin oil layer from the external

solution initiates target gene expression. The induced expression of the synthetic fluorescent proteins

from at least ∼100 bacteria per droplet generates detectable fluorescence signals to enable

fluorescence-activated cell sorting (FACS) of the intact droplets. This technology obviates time- and

labor-intensive cell culture typically required in conventional bulk experiment.

Introduction

For over sixty years, the tools to synthesize, manipulate andanalyze DNA have grown to encompass new extremes in bothscale and precision. Driven by miniaturization technologies,our ability to read and write DNA has improved dramaticallyover the last decade. High-throughput sequencing techno-

logies such as next-generation sequencing (NGS) have enabledthe analysis of many genetic and biochemical processes at anunprecedented scale and low cost.1,2 Emerging technologieson parallelized and miniaturized synthetic techniques to con-struct DNA sequences have led to a significant improvement inour ability to understand and engineer biology. Following theearly demonstrations of gene assembly using microarray-derived oligo pools,3–5 exciting developments have been madeto improve the quality and efficiency of microarray-based oligosynthesis and gene assembly.6–10 We have previously developeda microarray inkjet synthesizer to synthesize pools of thou-sands of codon-usage variants for protein expression optimiz-ation at low cost and high throughput.6

Despite the tremendous improvement in both DNA syn-thesis and sequencing, throughput and scale of the currentexperimental workflow in real practice remain limiting. This isdue to a bottleneck existing in the screening step where thedownstream cost of testing individual biological constructs forfunction is often far more expensive than the cost of synthesis.

†Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr08224f‡Present Address: Department of Mechanical Engineering, MassachusettsInstitute of Technology, Cambridge, USA, 02139.§These two authors contributed equally to this work.

aDepartment of Biomedical Engineering, Duke University, Durham, 27705, USA.

E-mail: kam.leong@columbia.edubDepartment of Biomedical Engineering, Columbia University, New York, 10027, USAcGeneral Biosystems, Inc. Morrisville, 27560 USAdTianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin,

300308, ChinaeDepartment of Systems Biology, Columbia University, New York, 10027, USA

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In addition, since the engineering information is encoded inthe genotype while the selection depends on the phenotype,this requires the genotype and phenotype to be linked in anyscreening strategy.

Cells are the most commonly used vehicles to bridge thisgenotype–phenotype linkage. Synthetic constructs are usuallyintroduced in recombinant forms and individual cells arepicked from culture plates and analyzed either by hand or byrobotic-pickers.6,11 The process is labor-intensive and time-con-suming. While eukaryotic cells screened by FACS can potentiallyaccess libraries larger than 108,12 flow cytometric analysis ofbacteria is still infrequent due to their small size and the rangeof screening is limited to survival or cell-bound products.6,13,14

Automation through the use of colony pickers increases thethroughput to 104 clones per day but cannot handle largerlibraries unless multiple machines are used in parallel.

In vitro compartmentalization (IVC) of individual elementsof the library in discrete, miniaturized W/O emulsions offersan attractive alternative for coupling genotype and phenotypeand simultaneously improves the cost-effectiveness andscreening sensitivity by reducing sample consumption andenhancing the signal response.15–17 Bulk emulsions sufferfrom polydispersity and the lack of control of reaction volume,timing and generality. These problems can be overcome byusing a droplet-based microfluidic system which allows theproduction of homogeneous and uniform droplets.18–24

However the W/O emulsions are incompatible with sustainedcell culture or any aqueous phase-based analysis (e.g. flow cyto-metry), as the immiscible oil phase is prone to evaporationand insoluble for polar nutrients typically.25

The problem can be circumvented by entrapping W/O dro-plets in another aqueous phase, forming W/O/W DE. Theexternal aqueous phase minimizes desiccation and enablesdroplet sorting via FACS. The middle oil shell functions as aselective barrier to regulate molecule transport, allowingsupply of nutrients or input of small inducer molecules, suchas anhydrotetracycline (aTC).24,26 Consequently, the dropletsare considered “programmable” in the sense that the innermicroenvironment can be modified by diffusion of moleculesinto the core to elicit a change in cell behavior. In this study,we present the development of a high-throughput screeningplatform for synthetic genes using a single bacterium encapsu-lated in microfluidics-generated DE droplets (Scheme 1). Theinducer molecule IPTG will be introduced in the externalphase to trigger gene expression upon its diffusion into thedroplet and the effect of its diffusion into the droplet on singlebacterium proliferation/signal amplification will be studied forthe first time.

Fluorescent protein genes were synthesized with a custom-built inkjet synthesizer, starting with oligos synthesis beforeundergoing isothermal oligonucleotide amplification and par-allel gene assembly, and inserted into E. coli cells for screen-

Scheme 1 Workflow of high-throughput screening of microchip-synthesized genes using double emulsion (DE) droplets. (A) In situ gene synthesisis performed on microchip using inkjet gene synthesizer. Oligos are first printed in microwells, after which enzymatic reactions take place to firstamplify and displace overlapping oligos from the original synthesized strand, and then assemble them via polymerase chain assembly to generatefull length DNA. (B) Synthetic genes are then cloned and transformed into E. coli. (C) Single bacterium is encapsulated into DE droplets before pro-liferation for signal enhancement. (D) High-throughput screening is achieved with FACS.

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ing. We then encapsulated an individual bacterium bearingindividual gene variants into DE droplets. The intrinsic limit-ation of chemical gene synthesis by stepwise addition of anucleotide monomer results in errors such as deletion andsubstitution.27 This is especially true for gene synthesis usingmicroarray-produced oligos, where error rates tend to behigher.3 The DE platform allows us to identify bacterial cloneswith functionally-correct rfp sequences. In addition, thediffusion of IPTG into the droplet can trigger the pET vector,under the control of a T7 promoter and a lac operator, regulat-ing the expression of the synthetic gfp (green fluorescentprotein) gene inserted into the bacteria. Since the pETexpression system is one of the most widely used systems forthe cloning and expression of synthetic and recombinant pro-teins in E. coli, the induction of pET expression by an externaltriggering mechanism offers an effective protocol to inducevarious target protein expressions for screening within the dro-plets. The capacity of the DE system (bacterial proliferationand activation of genes by chemical diffusion) combined withhigh-throughput sorting by FACS provide the basis for screen-ing complex gene libraries for a broad range of functionalityand activity.

ExperimentalMicrofluidics device fabrication

Microfluidic devices were fabricated by conventional soft litho-graphy techniques. A patterned silicon mold of 50 μm inheight and channel width was prepared from SU-8 2150(MicroChem, Newton, MA) according to the published proto-cols.26,28 A PDMS prepolymer and curing agent (Sylgard 184Silicone Elastomer Kit, Dow Corning, Midland, MI) were mixedat 10 : 1.05 mass ratio before being poured on top of thesilicon mold to cure at 80 °C for 1 hour. This ratio was chosenbased on our experience that the PDMS produced would haveoptimal stiffness for easy handling. A cover slide was bondedwith the device after holes at inlets and outlets were punchedand oxygen plasma treatment for 40 s at 20 W (Plasma Asher,Quorum Technologies, West Sussex, and RH). To create ahydrophilic surface along the channels, the devices werecoated following a two-step sol–gel coating procedure.26

DE droplet generation and characterization

DE droplets were generated as described in the main text.Bacterial culture medium was used as the inner aqueousphase. The oil phase used was HFE-7500 (Miller-StephensonChemical Co. Inc., Danbury, CT) supplemented with a Pico-Surf TM 1 surfactant (1%) (Dolomite Microfluidics,Charlestown, MA). The outer aqueous phase comprised aculture medium supplemented with Pluronic F-127 (2.5 wt%).The flow rates of the inner aqueous phase (1 μL min−1),the middle oil phase of HFE7500 (3 M, St Paul, MN)(2–7 μL min−1) and the outer aqueous phase (30–70 μL min−1)were controlled by using a Harvard Apparatus PHD 2000Syringe Pump.

Gene synthesis and error correction

The construction and error correction of synthetic fluorescentprotein genes were detailed in our previous publication.29

Briefly, after polymerase chain reaction (PCR) amplification ofthe on-chip assembled gene was completed, the gene productswere purified by agarose gel electrophoresis and extracted.They were melted by heating at 95 °C for 10 min, cooled to85 °C at 2 °C s−1 and held for 1 min. They were then cooleddown to 25 °C at a rate of 0.3 °C s−1, holding for 1 min at every10 °C interval. For ECR using a 20 min Surveyor cleavage incu-bation, 4 μl (200 ng) of the re-annealed gene product wasmixed with 0.5 μl of Surveyor nuclease and 0.5 μl enhancerand incubated at 42 °C for 20 min. Two μl of the reactionmixture was used for subsequent overlap extension-PCR(OE-PCR) using the same reaction conditions as the PCRdescribed above. The products were melted again for seconditeration following the same procedure.

Cloning of synthetic genes

Synthetic gene products (gfp, cyan fluorescent protein (cfp),and rfp) were cloned into a pAcGFP1 vector using a circularpolymerase extension cloning (CPEC).30,31

To prepare the pET expression plasmid, a synthetic gfp genewas inserted into a pET-28a(+) (Novagen Inc., Madison, WI,USA) vector containing a lacI gene, a T7 promoter, a lac oper-ator, and an ampicillin resistance gene. The cloning productwas transformed into BL21(DE3) chemically competent E. colicells (Invitrogen) according to the manufacturer’s instruction.The cells were grown on an agar plate with 50 μg ml−1 kanamy-cin for approximately 16 h.

Encapsulation of bacteria cells

Single colonies containing rfp, gfp and cfp genes were selectedfrom the previously prepared LB agar plate and transferredinto 200 mL M9 broth or diluted LB broth (1 : 1 LBmedium : PBS). The inoculated culture was then thoroughlymixed and diluted to reach the desired cell density (e.g. ∼106

cells per mL to obtain ∼0.06 cells per 60 pL droplet) beforebeing encapsulated into DE droplets. The flow rates of threephases (inner aqueous : middle oil : outer aqueous) were set at1 : 2 : 30 μL min−1 respectively. The droplets were collected andtransferred to 96-well plates containing M9 or diluted LBmedia for subsequent culture and analysis. >200 droplets wereimaged for determining the number of cell-containingdroplets.

IPTG induction of gene expression

Single colonies containing the pET-gfp plasmid were pickedfrom the LB agar plate and transferred into 200 mL M9 brothor diluted LB broth (1 : 1 LB medium : PBS) containing 50μg ml−1 kanamycin. A mixture of LB and PBS was used toreduce the effect of autofluorescence of LB. The inoculatedculture was then thoroughly mixed and diluted to reach thedesired cell density, and then encapsulated into DE droplets.The droplets were collected and transferred to 96-well plates

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containing 200 mL M9 or diluted LB media. IPTG was addedimmediately to the outer aqueous media to obtain a series ofconcentrations: 0 mM, 0.5 mM, 2 mM, 5 mM, 10 mM, 20 mMand 40 mM. The droplet fluorescence intensity was thenanalyzed at various time points with fluorescence microscopy.

Fluorescence microscopy

Droplets containing fluorescence-bearing bacteria were sus-pended in a 96-well plate and examined by using a NikonEclipse TE2000-U fluorescence inverted microscope at varioustime points following encapsulation. Fluorescence intensitywas analyzed by Image J. To compare fluorescence signalsemitted when different concentrations of IPTG were applied,all data points were normalized against the fluorescence signalmeasured at the first time point after 2 mM was applied exter-nally at t = 0.

Flow cytometry analysis

E. coli expressing GFP constitutively or under the control of apET expression vector were diluted in PBS buffer and encapsu-lated in the DE droplets. An equal number of droplets werethen suspended in PBS solution or M9 growth medium forcomparison of cell growth over time by flow cytometry(FACSCanto II, BD Biosciences, Franklin Lakes, NJ). The FSC/SSC was gated with empty droplets and free bacteria (negativecontrol) to specifically determine the population of dropletswith bacteria encapsulated. More than 10 000 droplets weremeasured each time to ensure reliable statistics. FlowJo (v.7.6,Tree Star, Ashland) was used to analyze the data.

Results and discussionIn situ gene synthesis from a custom array-based oligosynthesizer

We synthesized DNA microarrays using a custom-built inkjetDNA synthesizer for on-chip oligo amplification and geneassembly. A microchip made of a thermoplastic cyclic olefincopolymer (COC) was functionalized with hydrophilic SiO2

thin film arrays (Fig. S1†), creating physically isolated picoliter-sized reactors that constrained the liquid via differential wett-ability (Fig. 1A). These functionalized SiO2-COC microchipsexhibited enhanced droplet confinement and a reduced edge-effect during in situ DNA synthesis, producing high-qualityoligonucleotide arrays (Fig. 1B and S2†). Oligos were syn-thesized according to standard phosphoramidite chemistrybefore they were cleaved and assembled within isolated hybrid-ization chambers using combined nicking strand displace-ment and polymerase cycle assembly (nSDA–PCA) reaction(Fig. S3†). The formation of gene products was confirmed bygel electrophoresis of the PCR reaction products (Fig. 1C). Wechose gfp, rfp, and cfp as test genes for convenient screening offunctionally distinguishable genes, which served as an over-simplified model-library to assess the capability of the screen-ing platform. The gfp, rfp and cfp constructs were inserted intoa modified pAcGFP1 expression vector (by introducing a stopcodon within the multiple cloning site region so that the pre-inserted GFP sequence would not be expressed) using theCPEC cloning method.31 The recombinant products were thentransformed into bacteria, and fluorescent colonies wereselected and sequence-verified for each gene construct(Table S1†).

Fig. 1 (A) Scanning electron microscopy image of microchips (scale bar = 500 μm). (B) Fluorescein isothiocyanate solution constrained withinmicrowells via differential wettability. (C) Gel image showing microchip-synthesized green fluorescent protein (gfp), red fluorescent protein (rfp) andcyan fluorescent protein (cfp) genes.

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Single cell encapsulation and amplification in DE droplets

The high-throughput generation (>200 Hz) of picoliter-sizedDE droplets (∼60 pL & ∼50 μm in diameter) was carried out intwo polydimethylsiloxane (PDMS) flow-focusing devices con-nected serially (Fig. 2A): the first device produced W/O emul-sions, followed by a second device to supplement an outeraqueous phase to form W/O/W emulsions.32 The distributionof cells in a droplet follows Poisson distribution where theprobability of finding a droplet with k cells is defined byeqn (1):

f ðkÞ ¼ λke�λ

k!ð1Þ

where λ is the average cell number per droplet and k is thespecific cell number in the droplet.

To demonstrate single cell encapsulation and the sub-sequent population enrichment of bacteria in DE droplets,E. coli cells expressing synthetic gfp, rfp and cfp fluorescentproteins were loaded into 50 μm-diameter DE droplets(∼60 pL) at a density of ∼1 × 106 bacteria per mL, which pro-duced an average of <0.06 bacteria per droplet.

After 24 h of incubation, the proliferation of bacteria gener-ated distinctively red, green and cyan fluorescence signals inan individual droplet (Fig. 2B). No co-localization of differenttypes of bacteria was observed, indicative of the successful sep-aration of bacteria at the single cell level. Analysis over a largepool of droplets indicated a bacterial distribution matchingthe Poisson distribution of 0.01 bacteria per droplet on average(Fig. S4†). In this case, the probability of having two or more

bacteria per droplet was negligible; suggesting that such aloading cell density could effectively separate all bacteria intosingle cells per droplet.

When a DE droplet was used to encapsulate a single bacter-ium in growth medium (1 : 1 LB/PBS), the fluorescence inten-sity from the entire droplet was enriched by approximately 100times over a period of 24 h due to cell proliferation (Fig. 2Cand D). This observation confirms that culturing the bacter-ium inside the droplets allows both bacteria separation as wellas signal amplification from a single bacterium.

Screening of functional correctness for microarray-synthesizedand error-corrected genes

To determine how the screening system compared with theconventional culture plate method in terms of productivityand reliability, we applied the platform to estimate the errorfrequency of microarray-synthesized genes without prior selec-tion and sequence verification. We chose red fluorescentprotein (rfp) as a test gene for convenient screening of func-tionally-correct genes, which served as a good approximationof sequence-correct genes. In our previous publication, wereported that Surveyor nuclease, a commercialized form of theCEL endonuclease, was effective in removing errors duringchip-based gene synthesis.29 Freshly synthesized rfp gene con-structs before and after error-correction following our pre-viously reported protocol were transformed into E. coli cellswhich were then encapsulated into DE droplets as single cells.The resultant droplets were incubated in growth medium over-night to allow cell proliferation to saturate the inner droplet.

Fig. 2 (A) Schematic diagram of DE droplet generation and bright field images showing W/O and W/O/W droplets generated in 1st and 2nd chiprespectively. (B) Single cell encapsulation of bacteria carrying gfp, rfp & cfp genes showing no colocalization at 24 h post-encapsulation. (C) RelativeRFP intensity measured inside the droplets over time (n ≥ 10). (D) Fluorescence microscopy images showing the proliferation from a single RFP-expressing E. coli cell encapsulated in droplet.

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Using droplet counting, it was found that 52.8% of bacteria-positive droplets formed from uncorrected product containedcells that fluoresced brightly. The percentage of fluorescentcells approximated from the droplet system was consistentwith that calculated using colony counting on agar plates(Fig. 3A and B). Employing error correction increased the per-centage of brightly fluorescent RFP droplets to 90.6%, which isalso consistent with previous results obtained under conven-tional agar plate conditions.29

Tunable induction of synthetic gene expression through IPTGdiffusion into droplets

To investigate the potential application of microfluidics-gener-ated DE droplets as a perturbable microenvironment to screenand characterize synthetic gene expression, we first studied thefeasibility of inducible gene expression in the DE dropletthrough the diffusion of IPTG from the external aqueousphase. In the bulk environment, the expression of GFP inthese cells could be activated within a few hours by the appli-cation of IPTG. We encapsulated ∼30 BL21(DE3) E. coli cellscarrying a microarray-synthesized gfp controlled by both the T7promoter and lac operator in a pET vector in each droplet.Upon addition of 5 mM IPTG in the external aqueous phase,GFP expression became detectable after 4 h, but not in controldroplets without IPTG (Fig. 4A). The relative GFP intensityobserved per droplet increased over time, which was both con-tributed by increased GFP expression per cell (indicated bybrighter bacteria) and bacterial growth. The appearance of theGFP signal in the bacteria suggested effective transport of theIPTG molecule across the oil shell.

To further study the induction of GFP expression by IPTGdiffusion, we compared the relative fluorescence intensity (nor-malized against the intensity of bulk culture at 12 h) changeover time in droplets with that in the bulk culture environ-ment. Gene expression was delayed by about 4 h in dropletscompared with the conventional culture environment(Fig. 4B). To understand the impact of IPTG concentration on

gene expression induction, bacteria-containing droplets weresuspended in medium with a gradient of IPTG concentration.A concentration-dependent activation of GFP expression wasobserved (Fig. 4C). However, even at a low IPTG concentrationof 0.5 mM, the system was still able to achieve ∼80% of themaximum gene expression level obtained under higher con-centration conditions within 8 h of induction. This obser-vation confirms the relatively robust and efficient transport ofIPTG across the droplet shells.

We next investigated the effect of IPTG diffusion on geneexpression in droplets encapsulated with a single cell, andhow this process would interfere with bacterial proliferation.Bacteria encoding synthetic GFP in the pET vector were sus-pended in minimal medium (M9) and encapsulated into DEdroplets to yield no more than 1 bacterium cell per droplet.The M9 medium was chosen to optimize imaging conditionsdue to its low autofluorescence properties. The resultant dro-plets were cultured in medium containing a broad range ofIPTG concentrations from 0.5 to 40 mM.

As shown in Fig. 4D and E, an IPTG concentration above5 mM was inhibitive to the growth of bacteria. At 5 mM, theGFP intensity inside droplets fluctuated over time, suggestingthe inconsistent effect of inhibition of cell growth and geneactivation across the pool of droplets. Lower IPTG concen-trations, between 0.5 and 2 mM, were able to activate geneexpression under the control of a T7 promoter and lac operatorin pET vectors without interference with bacterial amplifica-tion. Noticeably upon onset, the collective GFP intensity waspositively correlated with the supplied IPTG concentration (0.5to 2 mM) with 2 mM IPTG providing the highest inducedexpression level. Cell populations at this point were relativelylow and uniform across different conditions. The collectiveGFP intensity was contributed mainly by gene expression inindividual cells. Yet this correlation gradually inverted itself ascells proliferated over time. At 12 h post-induction, dropletssupplemented with 1 mM IPTG started to exhibit a higher GFPintensity than that induced by 2 mM IPTG, whereas droplets

Fig. 3 Characterization of fluorescent cell population transfected with synthetic RFP gene before or after error correction. (A) Fluorescencemicroscopy images showing increased percentage of fluorescent droplets after error correction. Circled droplets contain bacteria that are eitherfluorescent (pink) or non-fluorescent (yellow). (B) Percentage of fluorescent clones was measured before and after error correction for RFP geneconstruct.

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activated by 0.5 mM IPTG generated the highest collective GFPintensity among all concentrations tested at the end point.This was partly due to the relatively faster growth of bacteriapost-induction than that observed when 2 mM to 40 mM IPTGwere applied, generating a large cell population encodinggreen fluorescent protein.

We next characterized and compared the appearance andmorphology of bacterial clusters for droplets in each IPTG con-centration (Fig. 4D). Besides generating the largest cell density

and in turn exhibiting the highest collective GFP intensity, asingle bacterium induced by 0.5 mM IPTG grew into stableand uniform cell clusters that saturated the entire inner phaseof the droplet. A small deviation of intensity was thus observedacross individual droplets under the same conditions.Nevertheless, as the IPTG concentration increased, the averagecell density dropped significantly while variation across dro-plets escalated within each condition, as indicated by the errorbar in Fig. 4E.

Fig. 4 (A) Fluorescence microscopy images of droplets containing bacteria in the absence of IPTG (top panel) and in the presence of 5 mM IPTG(bottom panel) (scale bar: 100 µm). (B) Relative fluorescence intensities of bacteria with pET vector controlling a gfp gene in bulk and in dropletsafter addition of 5 mM IPTG over time (n = 9). Data points were normalized against the intensity of bulk culture at 12 h. (C) Relative fluorescenceintensities of bacteria with pET vector controlling a gfp gene 8 h after introduction of various concentrations of IPTG. Data points were normalizedagainst the intensity after 5 mM was applied. (D) Fluorescence images showing GFP expressing cell clusters per droplet at 8 h, 12 h and 24 h timepoint post-IPTG induction. Images were taken at 20× magnification. (E) Average GFP intensity per droplet as a function of time with the externalconcentration of IPTG ranging from 0.5 mM to 40 mM (n = 9). (F) Fluorescence images showing GFP expressing cell clusters per droplet at 12 h and24 h time point after IPTG induction in minimal M9 vs. growth LB/PBS media. Images were taken at 20× magnification. (G) GFP intensity per dropletas a function of time upon IPTG induction in minimal M9 vs. growth LB/PBS media. (H) GFP intensity per droplet as a function of time with IPTGintroduced prior to encapsulation at various concentrations. Fluorescence intensity curves are compared to the condition with IPTG introducedfrom external aqueous environment (red line). All data points for (E), (G) & (H) were normalized against the fluorescence signal measured at the firsttime point after 2 mM was applied externally at t = 0.

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To further investigate the optimal induction conditions, weswitched to a more nutritious medium i.e. LB/PBS (1 : 1) in anattempt to boost bacterial metabolism and reduce the lag timeby facilitating the initial growth of bacteria using LB/PBS. Weencapsulated a single bacterium into DE droplets with eitherM9 or LB/PBS (1 : 1) media and suspended the droplets in 0.5or 2 mM IPTG culture. The fluorescence signal from bacteriacultured in LB/PBS broth increased exponentially after 4 hupon encapsulation, and was much higher at both 12 h and24 h time points than when M9 media was used (Fig. 4Fand G). This enhanced signal was partly due to a larger cellpopulation in each droplet. Particularly for 0.5 mM IPTGadded to the outer core, intensity achieved at 12 h in LB/PBSbroth outweighed that obtained from M9 broth at 24 h timepoint. Consistent with previous observation, a lower IPTG con-centration allowed single cells to proliferate into a saturatedcell density, resulting in a uniform and consistent signal ineach droplet. The cells induced by higher IPTG concentration,albeit being brighter themselves, generated an enhancedsignal variation due to the presence of a lower cell numberand dispersed cell pattern in each droplet. These observationssuggest that the IPTG diffusion rate and bacterial growth curvecould be synchronized to achieve the best signal amplificationeffect.

To further understand how IPTG would affect the growth ofa single bacterium in the droplet, we introduced a moderateamount of IPTG into bacterial culture prior to encapsulation.Single cells that came into direct contact with IPTG, even at anultralow concentration of 0.2 mM, proliferated at a much lower

rate than those which were activated by the IPTG slowlydiffused into the core from the external environment (Fig. 4H).This further confirms our previous note that IPTG was inhibi-tive to bacterial growth when they came into contact with thecell before they reached the lag phase. We also noticed thatthe effect was proportional to the concentration of IPTG in theenvironment.

Analysis of gene expression by fluorescence-activated high-throughput droplet sorting

The high capacity microfluidics-based droplet technologyrequires an automated, high-throughput screening system toprocess and sort a large spectrum of activities. DE droplets arecompatible with most flow cytometric analysis platforms. Inaddition to the throughput, the ability to precisely discrimi-nate among DE droplets based on their fluorescence and theiruniform size is crucial for accurate screening.

To demonstrate stringent fluorescent-activated sorting ofsynthetic genes in DE droplets, we encapsulated a mixture ofE. coli cells carrying synthetic GFP and RFP genes in an equi-valent amount into DE droplets as single cells. After overnightincubation, we suspended the droplets in PBS and analyzedthem with a flow cytometry sorter. As shown in the intensityhistograms, both GFP-positive and RFP-positive dropletsrevealed confined and distinctive peaks, representing strongand uniform signal intensities (Fig. 5A). With proper channelcompensation, droplets containing GFP and RFP expressingcells were sorted into different reservoirs.

Fig. 5 (A) Separation of double emulsion droplets containing GFP and RFP expressing cells. Top: Intensity histogram of GFP and RFP channels.Bottom: Overlay of green and red channels showing RFP+–GFP+ populations. (B) Flow cytometry analysis of double emulsion droplets loaded with4 different numbers of GFP-positive cells per droplet. Top: Overlay of signal intensities obtained for each condition respectively. Bottom: Analysis ofdroplet mixture containing all four species with 1, 10, 100 and 1000 bacteria per droplet.

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Interesting to note is that unlike conventional mammaliancells, where the Forward Scatter (FSC) vs. Side Scatter (SSC)value reveals the size and morphology information of thesample, DE droplets are transparent and thus do not generatesimilar scatter patterns when excited by the lasers. In con-trast, the forward and side scatter information observed herewas most likely generated from the spherical structure of thecell cluster confined by the inner droplet. The robust yet con-fined signal intensity measured during FACS indicated asufficient cell population in each droplet. This is consistentwith our previous observation that efficient signal amplifica-tion through cell proliferation could be readily achieved froma single copy of bacteria/genotype with this system. The con-centrated localization of data points from positive dropletson the FSC vs. SSC plot further confirms that the emulsiondroplets were highly uniform in terms of size and internalcomposition.

To demonstrate the importance of signal amplificationprior to FACS, we conducted a parallel analysis of multiple DEspecies present in the same sample. DE droplets encapsulatedwith four different densities of GFP-positive cells at 1, 10, 100,and 1000 bacteria per droplet were created and analyzed inFACS. We observed that both signal intensity and spatialresolution were enhanced with an increased average numberof cells present per droplet, as indicated by the decreased peakwidth yet increased peak height. Upon overlaying the intensityhistogram for all four emulsion conditions, peaks corres-ponding to droplets containing 100 and 1000 bacteria perdroplet were distinguishable from adjacent peaks (Fig. 5B). Tofurther validate the hypothesis, we mixed these four bacteria-loaded droplet samples at equal volumes and analyzed thejoint sample in a flow cytometer. As expected, only dropletsloaded with higher cell density could be accurately discrimi-nated, while droplets with lower cell population generatedfaint and broad signals that could not be resolved from eachother or from empty droplets.

These observations demonstrated the possibility of coup-ling FACS with a microfluidic DE system to achieve the desiredcapacity, throughput and automation of synthetic gene screen-ing. More importantly, these findings also highlight the sig-nificance of signal enrichment in stringent sorting duringFACS, as sufficient cell population is essential to generaterobust and uniform signals to guarantee detection sensitivityand resolution.

Discussion

In this study, we demonstrated the application of DE dropletsto encapsulate and culture a single bacterium carrying syn-thetic gene generated from a high-throughput microarray genesynthesizer. Coupling high-throughput gene synthesis with DEdroplet screening is especially advantageous when a knowngene library such as a library of codon-usage variants needs tobe screened for optimal protein expression.6 Efficient prolifer-ation of a single bacterium in DE droplets is critical in pheno-

type screening as a sufficient number of target molecules perunit volume of sample is essential to all biological detection. Asingle bacterium is difficult to be visualized under an opticalmicroscope as it measures around one micron in diameter andmoves rapidly at erratic trajectory. The signal intensity oftenfalls below the detection threshold of many screening techno-logies. Furthermore, the rapid and stochastic motion of indi-vidual cells in an aqueous environment causes them to period-ically gather at the center or diffuse to the periphery of thedroplet, creating signals that are temporally and spatiallyunstable. The lack of uniformity and stability of signals from aparticular genotype would greatly hinder the detection resolu-tion and sensitivity when phenotyping with high-throughputplatforms like FACS. Therefore, efficient enrichment of thebacterial population inside the droplet through self-replicationsignificantly amplifies the selective signals and enables strin-gent screening of enhanced protein expression of syntheticgenes. A saturated cell population confines the collectivemotion of the cells, allowing cell distribution to be spatiallyuniform and temporally static, which in turn increases boththe resolution and sensitivity of the screening process.

The proposed DE system is a tunable microenvironmentsuitable for single cell analysis as it allows transport of inducerchemicals to activate gene expression and enables efficientsignal amplification through robust cell proliferation. Theyield of single cell encapsulation is currently low (∼1% of dro-plets contained single cells while ∼99% were empty). Toincrease the yield, the use of a high aspect-ratio channel or acurved continuous microchannel has been proposed to over-come the limitation of stochastic cell loading which may beadopted in future work.33,34 The delayed-onset of IPTG-trig-gered gene activation through molecular diffusion can be fine-tuned to synchronize with the cell growth curve, facilitatingthe effective activation and amplification of synthetic genesfrom a single bacterium. The fine-tuning process depends onthe optimization of both the bacterial culture medium andIPTG concentration which resulted in the saturation of bac-teria in droplets and consequently the creation of a stable,spherical and fluorescence signal originating from the cellcluster. At high IPTG concentrations, the cells were most likelyoverburdened with plasmid expression, which diverts cellularresources from making necessary proteins for proliferationand leads to a reduction in the growth rate. This observation isconsistent with that reported in the literature.27,35 The inhi-bition of cell proliferation due to early exposure of bacteria toIPTG again highlighted the importance of diffusion of IPTGfrom the external aqueous phase into the droplet core toinduce gene expression. Analysis of droplets containingdifferent numbers of bacteria in FACS verified our hypothesisthat signal enhancement through bacterial proliferationincreases the resolution and sensitivity of the screeningprocess. Compared with conventional agar plate culture, ourproposed technology holds potential to replace manual count-ing and picking bacterial colony with automation, therebyadvancing the field of high-throughput synthetic genescreening.

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Conclusions

In conclusion, this study demonstrated the development of amicrofluidics-based platform that generated well-controlledmonodispersed DE droplets for high-throughput syntheticgene screening. We demonstrated single bacterium encapsula-tion in DE droplets, enabling the screening of functionally-correct genes (which gave an approximation of error-freegenes) generated from a microarray gene synthesizer beforeand after the error correction process. The diffusion of IPTGinto the droplet core induced gene expression, in which0.5 μM IPTG applied in the external phase resulted in the cre-ation of a stable, spherical fluorescence signal after 24 h.Fluorescence signals generated from at least 100 bacteria perdroplet were distinguishable in FACS, indicative of the impor-tance of signal amplification from a single bacterium in DEdroplets. The coupling of high-throughput gene synthesis andDE droplet screening system should open up opportunities toproduce and screen large amounts of synthetic genes efficien-tly. This study on single-cell encapsulation and IPTG-triggeredgene expression should pave the way for future research ofscreening complex gene libraries for a broad range of function-alities and activities.

Acknowledgements

Funding support from NIH (HL109442, AI096305, GM110494),the Guangdong Innovative and Entrepreneurial Research TeamProgram No. 2013S086, and the Global Research LaboratoryProgram (Korean NSF GRL; 2015032163) is acknowledged.HFC acknowledges fellowship support from the Sir EdwardYoude Memorial Fund Council (Hong Kong).

Notes and references

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