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Programming the group behaviors of bacterial communities with synthetic cellular communication

Programming the group behaviors of bacterial communities with synthetic cellular communication Synthetic biology is a newly emerged research discipline that focuses on the engineering of novel cellular behaviors and functionalities through the creation of artificial gene circuits. One important class of synthetic circuits currently under active development concerns the programming of bacterial cellular communication and collective population-scale behaviors. Because of the ubiquity of cell-cell interactions within bacterial communities, having an ability of engineering these circuits is vital to programming robust cellular behaviors. Here, we highlight recent advances in communication-based synthetic gene circuits by first discussing natural communication systems and then surveying various functional engineered circuits, including those for population density control, temporal synchronization, spatial organization, and ecosystem formation. We conclude by summarizing recent advances, outlining existing challenges, and discussing potential applications and future opportunities. Keywords: Synthetic biology; Gene circuits; Bacterial communities; Cellular communication; Collective behaviors; Dynamics Background provide novel diagnostic tools, enable economic produc- Synthetic biology is a newly emerged research discipline tion of therapeutics, and enable the design of novel treat- that focuses on the engineering of novel cellular behaviors ment strategies for various diseases including cancer, and functionalities. Since the launch of the field in 2000 metabolic disorders, and infectious diseases [28,29]. [1,2], a wide range of synthetic gene devices have been In the last few years, the advances of synthetic circuits created, including switches [3-9], oscillators [10-13], have been further expedited, empowered by recent break- memory elements [7,14,15], and communication modules throughs in genetic engineering techniques such as novel [13,16-18], as well as other electronics-inspired genetic DNA assembly [30-33] and genome editing tools [34-37], devices, such as digital logic gates [19-22], pulse gene- advances in methodologies including those for rational rators [23], and filters [24,25]. With designed cellular circuit design and optimization [38-40], and quick enrich- behaviors and functionalities, engineered circuits have ment of parts and elements [41,42]. As a result, synthetic been exploited to understand biological questions and biologists are now in a position to engineer desired cellu- to address various real-world problems [26]. The field has lar phenotypes in a larger, faster, and cheaper fashion. shown tremendous potential for biomedical, environmen- One important class of synthetic circuits that are under tal, and energy-related applications [27]. For example, to- active development concerns the programming of bac- wards biomedical applications, engineered genetic circuits terial cell-cell communication and the group behaviors of contribute to the understanding of disease mechanisms, communities [43-48]. Successful examples include gene constructs responsible for cellular density control [18], spatiotemporal patterning [13,16,49,50], and ecosystem * Correspondence: luting@illinois.edu Equal contributors formation [51,52]. The engineering of community-based Department of Bioengineering, University of Illinois at Urbana-Champaign, circuits is essential and invaluable towards the implemen- 1304 W Springfield Avenue, Urbana, IL 61801, USA tation of complex but robust cellular functionality because Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W Gregory Drive, Urbana, IL 61801, USA of the following reasons: First, although microbes are Full list of author information is available at the end of the article © 2014 Kong et al.; licensee Springer. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Kong et al. Bioresources and Bioprocessing 2014, 1:24 Page 2 of 11 http://www.bioresourcesbioprocessing.com/content/1/1/24 single cell organisms, they are present dominantly in the downstream genes (luxI and luxR in this case). A positive form of communities in nature and in live bodies, such feedback regulatory architecture arises here from the as biofilms [53,54] and the human microbiome [55,56]. self-activation of LuxI synthesis to facilitate the synchro- Second, microbial physiology and functionality are strongly nization of the cellular population. Similar to V. fischeri, correlated with their forms - for instance, bacterial anti- many other Gram-negative bacteria also possess QS sys- biotic resistance is distinct when cells are in planktonic tems, including the LasI/LasR and RhlI/RhlR systems forms and biofilm forms [57,58]. Third, recent advances in Pseudomonas aeruginosa [75], the CarI/CarR system in in the biotechnological industry have clearly shown that Erwinia carotovora [76], and the EsaI/EsaR system in microbial consortia may provide many compelling advan- Pantoea stewartii [77]. From an engineering perspective, tages in producing products of interest and controlling these QS systems can be decomposed into two separate fermentation processes [59,60]. modules with one for signal production and the other for We are thus motivated in this article to overview the signal detection and response - when engineered in differ- advances of synthetic gene circuits towards the program- ent cells, the two functional modules will confer commu- ming of bacterial cellular communication and community nications between the two cells as shown in Figure 1C. behaviors. We will first discuss basic communication In Gram-positive bacteria, modified oligopeptides often modules that confer cell-cell coordination in communities. serve as the signaling molecules for cellular communi- We will then overview various functional gene circuits cation with the cooperation of two-component systems. that enable the implementation of desired dynamic group One classic example of this type of system is the Agr behaviors, including those for population density control, system in Staphylococcus aureus (Figure 1B) [78]. Here, temporal synchronization, spatial organization, and eco- the auto-inducing peptide (AIP) precursor, encoded by the system formation. We will conclude by summarizing re- gene agrD, is modified on its thiolactone ring and cent advances and discussing existing challenges, potential exported by AgrB protein. Upon the binding of AIP with applications, and future opportunities. the transmembrane protein AgrC, the transcriptional fac- Although not discussed here, it is important to note tor AgrA inside the cell is phosphorylated and then acti- that there has been considerable progress in developing vated, which leads to the induction of the transcription of synthetic cellular communication in eukaryotes such as the downstream genes (agrB/D/C/A here). In addition to mammalian cells and yeast, which has been surveyed in the Agr system, there are many communication systems the literature [51,61,62]. based on auto-inducing peptides, such as the fsr system in Enterococcus faecalis [79], the Com system of Streptococ- Review cus pneumonia [80], and the nisRK system in Lactococcus Basic communication modules [70]. To program collective behaviors in Gram-positive Despite their species diversity, bacteria often utilize similar bacteria, a modular partition of those AI systems can thus signaling systems for the implementation of their group been exploited (Figure 1D). behaviors [63,64]. For instance, quorum sensing (QS) is Other than the QS and AIP systems that are primarily prevalent in bacteria for coordinating their group behav- present in intra-species communication, there are inter- iors such as bioluminescence [65], biofilm formation [66], species communication systems that coordinate cellular pathogenesis [67] and antibiotic synthesis [68-70]. behaviors over multiple bacterial species. One such ex- ample is the communication systems mediated by the uni- Bacterial communication via nonvolatile signaling molecules versal signaling molecule autoinducer-2 (AI-2), a furanosyl In Gram-negative bacteria, acyl-homoserine lactones borate diester synthesized by LuxS from S-adenosyl- (AHLs) are commonly used as QS molecules for intra- methionine and present in roughly half of all sequenced species communication. These molecules are composed bacterial genome [81,82]. Towards programmable behav- of a homoserine lactone ring with an acyl chain of C4 to iors in multiple bacterial species, AI-2 is hence an ideal C18 in length [71,72]. AHL molecules are synthesized by candidate for exploitation. the LuxI family synthases and detected by the correspond- ing LuxR-type receptors [73]. One canonical example of Bacterial communication via volatile and gas molecules this class of communication is the QS system discovered The adoption of nonvolatile molecules, such as AHLs and in the bioluminescent marine bacterium Vibrio fischeri AIs, as the broadcast signal enables cellular coordination [74]. As shown in Figure 1A, LuxI, the autoinducer syn- across various species. However, communications via thase, produces the AHL molecule 3OC HSL that can those molecules require the presence of the both sender diffuse freely across the cell membrane and accumulate and receiver species in the same liquid environments or in with the increase of cell density. Once the AHLs reach gel-like setting within a short distance to allow for dif- a threshold concentration, they form a complex with fusion of signaling molecules. Volatile molecules, in con- the LuxR receptor and activate the transcription of the trast, can diffuse through air and circumvent the need of P Kong et al. Bioresources and Bioprocessing 2014, 1:24 Page 3 of 11 http://www.bioresourcesbioprocessing.com/content/1/1/24 AIP (A) (B) AHL LuxR AgrA LuxR AgrA LuxI AgrA agrB agrD agrC agrA luxR luxI PP 2 luxR luxI (C) Senders Receivers AHL LuxR LuxI LuxR luxR luxI P P con con luxI AIP (D) Senders Receivers AgrA AgrA agrB agrD x agrA agrC con P P con Figure 1 Cellular communication in bacteria. (A) The LuxI/LuxR quorum sensing (QS) system in the Gram-negative bacterium Vibrio fischeri. The system consists of the genes luxI and luxR and the cognate promoter P and P . Its signaling molecule is the acyl-homoserine lactone luxI luxR (AHL) 3OC HSL. (B) The Agr QS system in the Gram-positive bacterium Staphylococcus aureus. It consists of the genes, agrD, agrB, agrC, and agrA and the cognate promoter P . The auto-inducing peptide (AIP) is the signaling molecule of the system. (C) An engineered communication module adapted from the wild-type LuxI/LuxR system in (A). By expressing luxI, the sender cell (left) produces the signal AHL that diffuses to the extracellular milieu and further into the receiver cell (right) to alter the expression of the downstream genes X. (D) A synthetic communication module built from the Agr system in (B). The sender cell (left) produces and secretes the signaling molecule AIP that is sensed by the receiver cell (right), resulting in the expression shift of the gene X in the receiver cell. physical mediating settings for signaling, allowing for cells (receiver) via air to trigger the expression of genes more versatile, rapid, and large-scale communications of controlled by the cognate acetaldehyde-inducible pro- communities. moters. Therefore, the sender cells produced a concentric Weber et al. recently established a communication gradient of acetaldehyde that induced the dose-dependent system that utilizes acetaldehyde as signaling molecules gene expression of the receiver cells with the expression [51]. In their study, a bacterial strain (sender) was engi- level defined by the distance between the sender and neered to constitutively express alcohol dehydrogenase receiver cells. (ADH), an enzyme that converts ethanol in the medium In another example, Hasty and colleagues constructed to acetaldehyde. Due to its low boiling point (21°C), ndh-2, a gene encoding NADH dehydrogenase II (mem- acetaldehyde volatized and was broadcast to neighboring brane-bound respiratory enzyme), into an Escherichia coli X Kong et al. Bioresources and Bioprocessing 2014, 1:24 Page 4 of 11 http://www.bioresourcesbioprocessing.com/content/1/1/24 strain to confer the production of hydrogen peroxide (A) (H O ) [83]. H O is a thermodynamically unstable chem- 2 2 2 2 ical compound and is able to enter neighboring cells quickly to alter their redox state and inactivate ArcAB, resulting in the shift of the activity of the corresponding LuxR AHL downstream genes. Through the exploitation of H O ,a 2 2 novel route of airborne signaling molecule was created for fast and large-scale colony coordination. ccdB luxI LuxI Other communication mechanisms LuxR In addition to the common signaling mechanisms discussed above, bacteria also exploit a wide range of alternative Cell death approaches for communications, such as quinolone signal luxR luxI [84], diffusible signal factor [85], cyclic dipeptide [86], con diketopiperazines [86,87], and others [88,89]. One such representative mode of signaling is the use of indole, an (B) aromatic heterocyclic organic compound that is produced AHL by over 85 species of Gram-positive and Gram-negative bacteria and used as an extracellular signal for global LuxR coordination of various bacterial species [90]. Although ccdA LuxI little of those mechanisms have been explored for luxI LuxR synthetic biology applications, the broad spectrum of signaling systems provides a rich reservoir for engineering luxI luxR multicellular functionality. lac/ara Dynamic group behaviors of bacterial communities via engineered communications Cell death ccdB Cellular communications enable the coordination of sin- lac gle cells by sending and sensing the states of individuals. Figure 2 Cellular density control enabled by engineered Inspired by this natural capability of bacteria, synthetic cellular communications. (A) A communication-based gene circuit biologists have developed a set of engineered bacterial that confers the auto-regulation of cellular population density. In this populations with their group behaviors programmed from system, a positive correlation between cell density and AHL concentration designed artificial cell-cell communications. is essential and was created by having the cells constitutively produce LuxI that catalyzes AHL synthesis. At a low density, cells survive and grow normally because the expression of the toxin gene ccdB is not activated Population density control by a low AHL level. In contrast, when cell density achieves a critical level, The first communication-based synthetic circuit was ccdB expression is triggered by accumulated AHL, causing cellular death. built by You et al. in 2004 with the goal of creating a The density-dependent cell death ensures an automatic control of total dynamic, autonomous regulation of the cell density of population density. (B) A synthetic gene circuit conferring an Allee an E. coli population [18]. As illustrated in Figure 2A, effect in an isogenic population. Rather than the toxin CcdB in (A), the antitoxin CcdA was correlated with cellular density via AHL the Lux system from V. fischeri was introduced to con- concentration. At a low cell density, the cells cannot survive because struct cell-cell communication and was coupled to cell of their production of the toxin CcdB. At a high density, the production survival and killing via the CcdA/B toxin system. Here, of the antitoxin CcdA is triggered to neutralize the toxic effects from the LuxI protein catalyzes the synthesis of a small, dif- CcdB, resulting in normal cell growth. fusible AHL signaling molecule, 3OC HSL, which accu- mulates in the extracellular milieu and the intracellular environment as the cell density increases. When cells state. Indeed, a stable cell density was maintained for reach a sufficient density, the AHL binds to LuxR and more than 30 h with the variation within less than 5% in forms the LuxR/AHL complex that activates the expres- the study. This density control circuit laid a foundation sion of the killer protein LacZα-CcdB, leading to cell for using cellular communications to program bacterial death. On the other hand, cell death can cause a reduc- communities, allowing the extension of the control of tion of total population density and hence the level of population dynamics to the engineering of more sophisti- AHL production, which in turn allows the population to cated synthetic ecosystems. recover after killing. The continuous production and deg- In a recent work, Smith et al. utilized the density control radation of AHL make the cell density approach a steady circuit constructed above to create an artificial Allee effect Kong et al. Bioresources and Bioprocessing 2014, 1:24 Page 5 of 11 http://www.bioresourcesbioprocessing.com/content/1/1/24 in E. coli populations [91]. The Allee effect is a biological (A) phenomenon characterized by a correlation between population density and the mean individual fitness of a population [92]. To create such an effect, a synthetic gene circuit was constructed to contain the LuxI/LuxR system and the CcdA/B toxin-antitoxin system (Figure 2B). In LuxR this setting, the expression of LuxR/LuxI and CcdB (killer) is under the control of P promoter, while CcdA lac/ara AiiA luxR (rescue) was regulated by the cell density-dependent P lux luxI con promoter. When IPTG induction is on, the cellular popu- GFP LuxR lation growth rate is negative if the initial cell density is aiiA less than the critical value (C ) at which CcdA expres- crit gfp sion is not activated. However, if the initial cell density is luxI above C , AHL activates the production of LuxR and crit further drives the production of CcdA which rescues the (B) population by inhibiting the toxicity of CcdB. An Allee effect population was thus established to have a negative fitness below a threshold of cell density but a positive LuxR fitness when the density is beyond the threshold. This study provided new implications of engineered cellular luxR AiiA communication for controlling invasive species and the P luxI con spread of infectious diseases. GFP LuxR aiiA Temporal synchronization NDH gfp Complex cellular behaviors, such as biofilm formation and host invasion, often require the temporal coordination and ndh collective action of cellular populations [93,94]. Towards luxI this need, engineered communications offer a powerful solution. In a recent study, Hasty and colleagues reported the de- Figure 3 Synchronization of genetic oscillations by velopment of an artificial gene circuit that synchronizes communication-based circuits. (A) A QS-based gene circuit the oscillation of gene expression in individual cells [13]. that synchronizes the oscillation of gene expression in an isogenic Figure 3A shows their circuit design based on the QS bacterial population. The oscillation is enabled by positive feedback, arising from the self-activation of AHL synthesis, and negative elements of V. fischeri (luxI, luxR)and Bacillus thurigensis feedback, mediated by the AHL-degrading gene aiiA. The coupling (aiiA). The AHL 3OC HSL, synthesized by LuxI, of the two feedback loops results in robust oscillations of gene binds to transcriptional factor LuxR to form a complex expression of an entire bacterial population (thousands of cells). (LuxR-AHL) that activates the expression of luxI,which (B) An advanced gene circuit modified from (A) that enables leads to a positive feedback loop in regulation. At the large-scale synchronization of oscillatory gene expression. In addition to the coupled positive and negative feedback in (A), an additional same time, the LuxR-AHL complex also activates the positive feedback loop is introduced by coupling the production of expression of aiiA, a gene encoding the AHL degradation thermally unstable H O by NADH dehydrogenase (ndh)withAHL 2 2 enzyme, which leads to a negative feedback loop in biosynthesis, leading to global oscillation synchronization of millions regulation. The dual positive and negative feedback of cells. loops drive the sustained oscillation of gene expression of individual cells, and in the meantime, the signaling molecule AHL confers the synchronization of individual Building on their success of the synchronized oscillator, oscillations. Using a custom-tailored microfluidic device, the same group further advanced to create a more sophis- the authors were, for the first time, able to establish ticated genetic network that is capable of synchronizing and tune synchronized oscillations of an entire cellular oscillatory gene expression of populations across multiple population (thousands of cells). Compared with the spatial scales [83]. As illustrated in Figure 3B, the resear- single cell oscillators developed by the same group [11] chers placed a copy of the ndh-2 gene, which encodes and other researchers [1,10], the engineered cellular NADH dehydrogenase II, under the control of an add- communication indeed conferred the synchrony of cellu- itional copy of P promoter (compared with Figure 3A). lux lar gene expression dynamics at a robust and yet tunable The NDH-2 produces a low level of H O that vapors and 2 2 fashion. passes through the walls of the oxygen-permeable LuxI AHL LuxI AHL H O 2 2 Kong et al. Bioresources and Bioprocessing 2014, 1:24 Page 6 of 11 http://www.bioresourcesbioprocessing.com/content/1/1/24 polydimethylsiloxane (PDMS) chips. Driven by the oscilla- organs, or even entire organisms, one critical step is to tion of gene expression mediated by the AHL-based syn- develop an engineering strategy that enables robust spa- chronized oscillation circuit, H O was periodically tiotemporal pattern formation of living cells. Engineered 2 2 produced and exchanged between the cells within individ- cellular communications hold a great promise towards ual chambers. When entering cells, H O changes the this goal, in addition to their roles in conferring temporal 2 2 redox state of the cells and inactivates their lux promoter coordination of cellular behaviors. binding protein ArcAB, causing the global activation of In fact, synthetic biologists have already made several the lux promoter of the cells in different chambers. As a interesting attempts through the exploitation of artificial result, thousands of oscillating colony ‘biopixels’ (appro- communication-based gene circuits. For instance, Sohka ximately 2.5 million cells) were synchronized over et al. constructed a circuit implementing Wolpert's French centimeter-length scales through the use of synergistic flag model [96], enabling the determination of cell fates in intercellular coupling involving both quorum sensing a concentration-dependent manner [25]; Payne et al. cre- within a colony and gas-phase redox signaling between ated a circuit that allows self-organized pattern formation colonies. As a proof-of-concept application, this system without morphogen gradients in bacteria [97]; Basu et al. was further employed to sense arsenic in environments engineered a band detector that allows for differential via differential modulations of the period of the oscillatory response of gene expression according to the local cells that resemble a liquid crystal display (LCD)-like concentration of AHL, creating a bull's eye-like spatio- macroscopic clock. temporal pattern [16]. One elegant example for this line of applications is the Spatial organization programming of bacterial stripe patterns by Liu and co- One of the most fascinating aspects of biological systems workers [49]. As shown in Figure 4A, the gene circuit is their ability to generate complex but highly reproducible consists of two functional parts: density-sensing module organisms through differential spatial patterning of mor- and motility-control module. The density-sensing module phogens across isogenic cells [95]. Towards the ultimate centers on the LuxI/LuxR QS system that enables the syn- goal of biological engineering for creating desired tissues, thesis and excretion of the AHL and the activation of the (A) (B) Red light EnvZ OmpC LuxR AHL P LuxR luxR AHL con cI PCB cph8 LuxR luxI LuxR LuxI con cI LuxI cI Z CheZ pcyA cI ho1 luxI lacZ luxR cheZ luxI P P con ompC lux-lambda lambda con Figure 4 Spatial organization of cellular populations via engineered communication circuits. (A) A genetic circuit that generates periodic stripes in space. The LuxI/LuxR QS system is coupled to cellular motility via the transcriptional repressor gene cI, which is induced by AHL via the promoter P and inhibits the expression of cheZ, one of the essential genes in bacteria motility. At a low cell density, AHL concentration remains low luxI and no CI is produced, leading to a constant production of CheZ and hence a high cell motility. In a high cell density, sufficient accumulation of the AHL induces CI production which in turn suppresses cheZ expression, resulting in a deficiency in cell motility. The density-dependent motility of the population generates periodic stripe patterns in an expanding cell population. (B) A multi-module, communication-based synthetic circuit that allows accurate edge detection. Three functional modules are involved, including a light sensor, a cell communication module, and an X AND (NOT Y) gate. Upon exposure to red light, the light-sensing protein Cph8 induces the expression of cI and luxI: CI represses the expression of lacZ in the same cell regardless of AHL concentration while LuxI triggers the production of AHL that can diffuse to neighboring cells in the dark region to induce the production of LacZ. On the other hand, cells far from the light cannot produce LacZ because no AHL is available to trigger the transcription. As a result, only the cells near the edge of the light-exposed area actively express lacZ, which results in a dark pigment due to the enzymatic cleavage of a substrate in the plate by LacZ. Kong et al. Bioresources and Bioprocessing 2014, 1:24 Page 7 of 11 http://www.bioresourcesbioprocessing.com/content/1/1/24 downstream gene cI when cell density is sufficiently high. Figure 5A shows the design of the ecosystem that The motility-control module is based on the bacterial involves two QS modules, LuxI/LuxR from V. fischeri and motility system that is regulated by the transcription of LasI/LasR from P. aeruginosa, for two-way communi- cheZ. Upon the replacement of the wild-type cheZ cations. The predator cell (top) produces and secretes the with an inducible version (cheZ is under the control of the AHL 3OC HSL that induces the expression of the toxin cI-repressed P promoter), cellular motility becomes gene ccdB in the prey cell (bottom), leading to the death lambda regulated by the expression of cI. With the coupling of the of the prey. In the meantime, the prey produces another two modules, engineered E. coli populations were able AHL molecule, 3OC HSL, which rescues the predator by to form robust but tunable periodic stripes of high and inducing the production of antitoxin CcdA that neutral- low cell densities sequentially and autonomously. These izes the toxin from CcdB. With appropriate modulations results established cellular motility as a simple route to of the system parameters, the researchers were able to create recurrent spatial structures without the need for create a bacterial version of predation with different an extrinsic pacemaker. As a novel mechanism, it offered population dynamics generated, including extinction, an alternative solution for the formation of biological coexistence, and oscillation. Similar to this work, another spatial patterns that is distinct from the well-acknowledged bidirectional intercellular communication network was Turing mechanism [98]. also engineered by Brenner et al. [50], in which the LasI/ In addition to autonomous pattern formation, the LasR and RhlI/RhlR QS systems from P. aeruginosa were QS-based communication mechanism can also be applied adopted to create a two-species microbial consensus to detect complex spatial signals. Tabor et al. recently consortium. In that ecosystem, the gene expression of developed a multi-module gene circuit system for edge any of the two species mutually depends on the presence detection, a signal processing algorithm common in artifi- of the other. cial intelligence and image recognition [99]. As illustrated Beyond predation and consensus, designer cellular in Figure 4B, the biological edge detection algorithm is communications can be used to create a wide spectrum composed of three modules: a dark sensor (NOT light), of inter-species interactions. As revealed by metagenomics cell-cell communication cassette, and an X AND (NOT Y) and 16S pyrosequencing, microbial interactions in nature genetic logic. The darker sensor was engineered based on such as biofilms and the microbiome are extremely com- the light-sensitive protein Cph8, a chimeric sensor kinase. plicated and diverse - for instance, there can be parasitism, With the covalent association of chromophore phycocya- predation, commensalism, mutualism, competition, and nobilin produced from heme via ho1 and pcyA [100,101], amensalism within a single pair of species [102]. As one of Cph8 is able to activate the ompC promoter (P )by the earliest efforts towards the programming of com- ompC transferring a phosphoryl group to the response regulator plicated cellular consortia, Weber and Fussenegger devel- OmpR. However, in the presence of red light, the kinase oped a set of pairwise interactions between E. coli and activity of Cph8 is inhibited, which precludes the tran- Chinese hamster ovary (CHO) cells [51]. scription from P and causes a NOT light trans- As illustrated in Figure 5B, the designs of the ecosystems ompC criptional logic gate. The cell-cell communication was center on an airborne transmission of the transcription sys- implemented through the Lux QS system and was used to temthatallowsone species(E. coli) to convert ethanol into convert light information into spatial distribution of AHL. volatile acetaldehyde and broadcast this airborne signal With the incorporation of the converter cI and the hybrid (boiling point: 21°C) to another species (CHO-K1 cell line) promoter P ,the stateofP is converted via an for the activation of functionally specific, rationally engi- lux-lambda ompC X AND (NOT Y) logical operation into the state of the neered genes. The commensal ecosystem (top) was created promoter P , which is displayed via the production by constructing an E. coli strain capable of converting lux-lambda of LacZ that produces black pigment. Upon the loading of ethanol into acetaldehyde for air broadcast and placing a the programs, a lawn of isogenic E. coli populations was neomycin resistance gene (neo) under the control of an able to sense an image of light, communicate to identify acetaldehyde-induced promoter (P ) in a CHO-K1 cell air the light-dark edges, and visually present the result of the line. In addition, secreted alkaline phosphatase (SEAP) computation. was used as a reporter of the CHO-K1 cells. When cul- tivated proximate to synthetic CHO-K1, the engineered Ecosystem formation E. coli cells confer survival of the mammalian cells while Artificial cellular communications can enable not only keeping their own growth unaffected by the mammalian the coordination of isogenic cell populations but also cells cultured in a separate dish. The amensal ecosystem heterogeneous ecosystems that are composed of multiple (middle) was synthesized by cultivating an acetaldehyde- species. You and co-works recently developed two gene broadcasting E. coli strain in close proximity to a CHO-K1 circuits into a predator-prey ecosystem that consists of cell line that was engineered to have acetaldehyde- two E. coli populations [52]. controlled expression of RipDD, a gene that encodes an Kong et al. Bioresources and Bioprocessing 2014, 1:24 Page 8 of 11 http://www.bioresourcesbioprocessing.com/content/1/1/24 (A) (B) (C) Commensalism Culture well 2 Neomycin Culture O Cell death Initial colonizer cell Predator well 1 AlcR Cell growth + EtOH P NEO air CP25 Disperse lasR rfp A E. coli neo ccdB O SEAP lac/ara-1 P SV40 LtetO-1 seap BdcAE lasI luxR LasR ccdA RFP 50Q luxI LasR LuxR bdcAE50Q LasI LuxR Amensalism lasI Culture well 2 Culture O well 1 3OC HSL 6 3OC HSL O Cell growth 12 + EtOH AlcR Apoptosis E. coli 3OC HSL Disperser cell RipDD Prey 12 Disperse ripDD Cell death P air LasR Hha GFP LasI B 13D6 LuxI LasR Mutualism Culture well 2 Neomycin gfp lasI hha13D6 Culture O luxI lasR ccdB CP25 P T5-lac P P well 1 luxI lac/ara-1 AlcR Cell growth + EtOH air sBLA neo E. coli SEAP NEO SV40 seap Ampicilin Figure 5 Programmed ecosystems developed from designed cellular communications. (A) A synthetic predator-prey ecosystem in E. coli. The predator cell (top) produces the QS signal 3OC HSL that induces the expression of the toxin gene ccdB in the prey cell (bottom) and causes cell death. Meanwhile, the prey produces another QS signal, 3OC HSL, which triggers the expression of ccdA, an antitoxin gene whose expression rescues the predator by neutralizing the toxin CcdB accumulated inside the cell. (B) Synthetic ecosystems with E. coli and Chinese hamster ovary (CHO) cells. The three ecosystems were based on the same foundation - an airborne transmission of transcription system, through which the sender (E. coli) converts ethanol into volatile acetaldehyde and broadcasts it to the receiver (CHO-K1) to alter corresponding gene expression. Top panel: The volatile acetaldehyde produced by E. coli induces antibiotic resistance in CHO-K1 cells, leading to a commensal community. Middle panel: The acetaldehyde from the sender induces the apoptosis of the receiver, creating amensalism between the two species. Bottom panel: E. coli rescues the CHO-K1 cell by triggering antibiotic resistance through volatile acetaldehyde, and in the meantime, the CHO-K1 cell benefits E. coli by degrading ampicillin that is toxic to E. coli, resulting in a mutualistic consortium. (C) An engineered biofilm-forming system that consists of two communicating E. coli species. The disperser cell (bottom) produces AHL (3OC HSL) to trigger the expression of the gene bdcAB50Q in the initial colonizer cell (top), leading to the dispersion of the biofilm formed by the initial colonizer cells. Meanwhile, the biofilm formed by the disperser cells can also be dispersed by inducing the expression of the dispersal protein Hha13D6 with external inducer IPTG. The combination of the two steps allows the replacement and removal of biofilms in a programmed manner. apoptosis-inducing human receptor interacting protein. understanding and programming microbial community As a result, the CHO-K1 cells survive only in the absence patterns that orchestrate the complex coexistence of living of the E. coli cells because, otherwise, they induce the systems. death of the CHO-K1 cells by producing acetaldehyde. In addition to programming planktonic bacterial popu- To create a mutualistic interaction between E. coli and lations, synthetic communication circuits have also been CHO-K1 cells (bottom), the commensal ecosystem devel- exploited in controlling complex communities such as oped earlier (top) was modified to incorporate a mamma- biofilms. Hong et al. recently developed quorum-sensing lian beta-lactamase gene sBLA under the control of the circuits to program the formation and dispersal of acetaldehyde-inducible promoter (P ). Here, sBLA can be artificial E. coli biofilms [103]. As shown in Figure 5C, the air secreted to the extracellular milieu to hydrolyze the circuits have two functional parts with one belonging to bacterial antibiotic ampicillin in the culture medium the initial colonizer cell (top) and the other belonging to to promote the survival of co-cultured E. coli,resulting the disperser cell (bottom). The initial colonizer part in bidirectional benefits between the two cell species. consists of the constitutively expressed repressor gene Following a similar idea, three additional types of eco- lasR and its cognate promoter P that drives the expres- lasI system interactions were created, including parasitism, sion of the biofilm dispersion gene bdcAB50Q;the third party-inducible parasitism, and predator-prey inter- disperser part is composed of the AHL-producing gene action (not shown in Figure 5). This example demon- lasI that is constitutively expressed and another biofilm strated the ability of programming microbial consortia via dispersion gene, hha13D6, controlled by external inducer rational design of cellular interactions by rewiring cellular IPTG. Such a design allows the disperser cell to trigger communication systems, providing novel insights in the expression of the gene bdcAB50Q in the initial SBLA Kong et al. Bioresources and Bioprocessing 2014, 1:24 Page 9 of 11 http://www.bioresourcesbioprocessing.com/content/1/1/24 colonizer cell by producing AHL (3OC HSL), leading to productions [115-117]. There are a variety of research the dispersion and replacement of the biofilm formed by fields where synthetic bacterial consortia have started to the initial colonizer cells. Meanwhile, the circuit in the play an important role: In metabolic engineering, cellular disperser enables the biofilm formed by the dispersers to communication can be used to implement self-regulated be removed with the external signal inducer IPTG. These control between cellular growth and product manufac- types of functional circuits can be powerful in creating turing in bioreactors for autonomous bioproduction. In designer biofilms and enabling precise manipulation of biomedical applications, custom-tailored probiotic bac- community composition in the fields of biorefinery, teria can be introduced into the human body to alter the medicine, and bioproduction. composition and hence the function of the gut microbiota for disease treatment. In areas relating to the environ- Conclusions ment, biofilms and microbial consortia in soil and other With the advances of synthetic biology technologies and a natural settings can be perturbed and even reprogrammed consensus on the need for community-based functionality with engineered microbes for desired purposes. We thus engineering, synthetic microbial consortia have undergone expect that microbial communities programmed via a rapid development in the past few years. This review has engineered cellular communication will become a versa- surveyed recent advances of engineered biological systems tile strategy in addressing both scientific and practical that utilize cell-cell communication to program bacterial challenges in the near future. group behaviors, covering both the basic communication Abbreviations modules and functional gene circuits that confer desired ADH: alcohol dehydrogenase; AHL: acyl-homoserine lactones; AI-2: autoinducer-2; community-based dynamic behaviors. AIP: auto-inducing peptide; CHO cells: Chinese hamster ovary cells; Although there has been significant progress, the en- H O : hydrogen peroxide; Ndh-2: NADH dehydrogenase II; QS: quorum 2 2 sensing; SEAP: secreted alkaline phosphatase. gineering of microbial communities is still in its infancy and is subject to a set of challenges. In fact, almost all Competing interests synthetic circuits to date have involved many rounds of The authors declare that they have no competing interests. trial and error before achieving the desired functionality. Difficulties in the efficient construction of engineered Authors' contributions circuits often stem from a lack of biological knowledge. QH and TL conceived the study and designed the project. WT, VC, and TL drafted the manuscript. CL analyzed the data. All authors read and approved Specifically, to facilitate gene circuit engineering, it is the final manuscript. needed to have a deep understanding of stochasticity in gene expression [104-106], the inherent interplay Acknowledgements between a synthetic circuit and the host organism [1], We thank Andrew Blanchard for commenting and editing the manuscript. This work was supported by the American Heart Association (Grant No. and issues related to multicellular physiology and metab- 12SDG12090025), the Network for Computational Nanotechnology at UIUC olism [107]. Another big challenge arises from the tech- sponsored by National Science Foundation (Grant No. 1227034), and the nical side of synthetic biology, which includes the lack of UIUC Research Board. powerful rational design platforms, limited availability of Author details parts and modules, efficient systematic optimization strat- Department of Bioengineering, University of Illinois at Urbana-Champaign, egies and toolkits, and high-throughput assays for circuit 1304 W Springfield Avenue, Urbana, IL 61801, USA. Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W Gregory Drive, validation. Addressing the above challenges will foster our Urbana, IL 61801, USA. State Key Laboratory of Bioreactor Engineering, East engineering capability and help to achieve the ultimate China University of Science and Technology, 130 Meilong Road, Shanghai goal of efficient and reliable development of synthetic 200237, People’s Republic of China. Department of Physics, University of Illinois at Urbana-Champaign, 1110 W Green Street, Urbana, IL 61801, USA. circuits with defined functionality. Despite the challenges, the future of engineered micro- Received: 26 August 2014 Accepted: 21 October 2014 bial communities is bright. In fact, synthetic consortia have already started to show tremendous potential in both understanding biological questions and addressing References 1. Elowitz MB, Leibler S (2000) A synthetic oscillatory network of transcriptional real-world concerns. For example, extended from the regulators. Nature 403(6767):335–338 programming of cellular dynamics, synthetic bacterial 2. Gardner TS, Cantor CR, Collins JJ (2000) Construction of a genetic toggle systems have been applied to understand ecological and switch in Escherichia coli. Nature 403(6767):339–342 3. 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Programming the group behaviors of bacterial communities with synthetic cellular communication

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Abstract

Synthetic biology is a newly emerged research discipline that focuses on the engineering of novel cellular behaviors and functionalities through the creation of artificial gene circuits. One important class of synthetic circuits currently under active development concerns the programming of bacterial cellular communication and collective population-scale behaviors. Because of the ubiquity of cell-cell interactions within bacterial communities, having an ability of engineering these circuits is vital to programming robust cellular behaviors. Here, we highlight recent advances in communication-based synthetic gene circuits by first discussing natural communication systems and then surveying various functional engineered circuits, including those for population density control, temporal synchronization, spatial organization, and ecosystem formation. We conclude by summarizing recent advances, outlining existing challenges, and discussing potential applications and future opportunities. Keywords: Synthetic biology; Gene circuits; Bacterial communities; Cellular communication; Collective behaviors; Dynamics Background provide novel diagnostic tools, enable economic produc- Synthetic biology is a newly emerged research discipline tion of therapeutics, and enable the design of novel treat- that focuses on the engineering of novel cellular behaviors ment strategies for various diseases including cancer, and functionalities. Since the launch of the field in 2000 metabolic disorders, and infectious diseases [28,29]. [1,2], a wide range of synthetic gene devices have been In the last few years, the advances of synthetic circuits created, including switches [3-9], oscillators [10-13], have been further expedited, empowered by recent break- memory elements [7,14,15], and communication modules throughs in genetic engineering techniques such as novel [13,16-18], as well as other electronics-inspired genetic DNA assembly [30-33] and genome editing tools [34-37], devices, such as digital logic gates [19-22], pulse gene- advances in methodologies including those for rational rators [23], and filters [24,25]. With designed cellular circuit design and optimization [38-40], and quick enrich- behaviors and functionalities, engineered circuits have ment of parts and elements [41,42]. As a result, synthetic been exploited to understand biological questions and biologists are now in a position to engineer desired cellu- to address various real-world problems [26]. The field has lar phenotypes in a larger, faster, and cheaper fashion. shown tremendous potential for biomedical, environmen- One important class of synthetic circuits that are under tal, and energy-related applications [27]. For example, to- active development concerns the programming of bac- wards biomedical applications, engineered genetic circuits terial cell-cell communication and the group behaviors of contribute to the understanding of disease mechanisms, communities [43-48]. Successful examples include gene constructs responsible for cellular density control [18], spatiotemporal patterning [13,16,49,50], and ecosystem * Correspondence: luting@illinois.edu Equal contributors formation [51,52]. The engineering of community-based Department of Bioengineering, University of Illinois at Urbana-Champaign, circuits is essential and invaluable towards the implemen- 1304 W Springfield Avenue, Urbana, IL 61801, USA tation of complex but robust cellular functionality because Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W Gregory Drive, Urbana, IL 61801, USA of the following reasons: First, although microbes are Full list of author information is available at the end of the article © 2014 Kong et al.; licensee Springer. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Kong et al. Bioresources and Bioprocessing 2014, 1:24 Page 2 of 11 http://www.bioresourcesbioprocessing.com/content/1/1/24 single cell organisms, they are present dominantly in the downstream genes (luxI and luxR in this case). A positive form of communities in nature and in live bodies, such feedback regulatory architecture arises here from the as biofilms [53,54] and the human microbiome [55,56]. self-activation of LuxI synthesis to facilitate the synchro- Second, microbial physiology and functionality are strongly nization of the cellular population. Similar to V. fischeri, correlated with their forms - for instance, bacterial anti- many other Gram-negative bacteria also possess QS sys- biotic resistance is distinct when cells are in planktonic tems, including the LasI/LasR and RhlI/RhlR systems forms and biofilm forms [57,58]. Third, recent advances in Pseudomonas aeruginosa [75], the CarI/CarR system in in the biotechnological industry have clearly shown that Erwinia carotovora [76], and the EsaI/EsaR system in microbial consortia may provide many compelling advan- Pantoea stewartii [77]. From an engineering perspective, tages in producing products of interest and controlling these QS systems can be decomposed into two separate fermentation processes [59,60]. modules with one for signal production and the other for We are thus motivated in this article to overview the signal detection and response - when engineered in differ- advances of synthetic gene circuits towards the program- ent cells, the two functional modules will confer commu- ming of bacterial cellular communication and community nications between the two cells as shown in Figure 1C. behaviors. We will first discuss basic communication In Gram-positive bacteria, modified oligopeptides often modules that confer cell-cell coordination in communities. serve as the signaling molecules for cellular communi- We will then overview various functional gene circuits cation with the cooperation of two-component systems. that enable the implementation of desired dynamic group One classic example of this type of system is the Agr behaviors, including those for population density control, system in Staphylococcus aureus (Figure 1B) [78]. Here, temporal synchronization, spatial organization, and eco- the auto-inducing peptide (AIP) precursor, encoded by the system formation. We will conclude by summarizing re- gene agrD, is modified on its thiolactone ring and cent advances and discussing existing challenges, potential exported by AgrB protein. Upon the binding of AIP with applications, and future opportunities. the transmembrane protein AgrC, the transcriptional fac- Although not discussed here, it is important to note tor AgrA inside the cell is phosphorylated and then acti- that there has been considerable progress in developing vated, which leads to the induction of the transcription of synthetic cellular communication in eukaryotes such as the downstream genes (agrB/D/C/A here). In addition to mammalian cells and yeast, which has been surveyed in the Agr system, there are many communication systems the literature [51,61,62]. based on auto-inducing peptides, such as the fsr system in Enterococcus faecalis [79], the Com system of Streptococ- Review cus pneumonia [80], and the nisRK system in Lactococcus Basic communication modules [70]. To program collective behaviors in Gram-positive Despite their species diversity, bacteria often utilize similar bacteria, a modular partition of those AI systems can thus signaling systems for the implementation of their group been exploited (Figure 1D). behaviors [63,64]. For instance, quorum sensing (QS) is Other than the QS and AIP systems that are primarily prevalent in bacteria for coordinating their group behav- present in intra-species communication, there are inter- iors such as bioluminescence [65], biofilm formation [66], species communication systems that coordinate cellular pathogenesis [67] and antibiotic synthesis [68-70]. behaviors over multiple bacterial species. One such ex- ample is the communication systems mediated by the uni- Bacterial communication via nonvolatile signaling molecules versal signaling molecule autoinducer-2 (AI-2), a furanosyl In Gram-negative bacteria, acyl-homoserine lactones borate diester synthesized by LuxS from S-adenosyl- (AHLs) are commonly used as QS molecules for intra- methionine and present in roughly half of all sequenced species communication. These molecules are composed bacterial genome [81,82]. Towards programmable behav- of a homoserine lactone ring with an acyl chain of C4 to iors in multiple bacterial species, AI-2 is hence an ideal C18 in length [71,72]. AHL molecules are synthesized by candidate for exploitation. the LuxI family synthases and detected by the correspond- ing LuxR-type receptors [73]. One canonical example of Bacterial communication via volatile and gas molecules this class of communication is the QS system discovered The adoption of nonvolatile molecules, such as AHLs and in the bioluminescent marine bacterium Vibrio fischeri AIs, as the broadcast signal enables cellular coordination [74]. As shown in Figure 1A, LuxI, the autoinducer syn- across various species. However, communications via thase, produces the AHL molecule 3OC HSL that can those molecules require the presence of the both sender diffuse freely across the cell membrane and accumulate and receiver species in the same liquid environments or in with the increase of cell density. Once the AHLs reach gel-like setting within a short distance to allow for dif- a threshold concentration, they form a complex with fusion of signaling molecules. Volatile molecules, in con- the LuxR receptor and activate the transcription of the trast, can diffuse through air and circumvent the need of P Kong et al. Bioresources and Bioprocessing 2014, 1:24 Page 3 of 11 http://www.bioresourcesbioprocessing.com/content/1/1/24 AIP (A) (B) AHL LuxR AgrA LuxR AgrA LuxI AgrA agrB agrD agrC agrA luxR luxI PP 2 luxR luxI (C) Senders Receivers AHL LuxR LuxI LuxR luxR luxI P P con con luxI AIP (D) Senders Receivers AgrA AgrA agrB agrD x agrA agrC con P P con Figure 1 Cellular communication in bacteria. (A) The LuxI/LuxR quorum sensing (QS) system in the Gram-negative bacterium Vibrio fischeri. The system consists of the genes luxI and luxR and the cognate promoter P and P . Its signaling molecule is the acyl-homoserine lactone luxI luxR (AHL) 3OC HSL. (B) The Agr QS system in the Gram-positive bacterium Staphylococcus aureus. It consists of the genes, agrD, agrB, agrC, and agrA and the cognate promoter P . The auto-inducing peptide (AIP) is the signaling molecule of the system. (C) An engineered communication module adapted from the wild-type LuxI/LuxR system in (A). By expressing luxI, the sender cell (left) produces the signal AHL that diffuses to the extracellular milieu and further into the receiver cell (right) to alter the expression of the downstream genes X. (D) A synthetic communication module built from the Agr system in (B). The sender cell (left) produces and secretes the signaling molecule AIP that is sensed by the receiver cell (right), resulting in the expression shift of the gene X in the receiver cell. physical mediating settings for signaling, allowing for cells (receiver) via air to trigger the expression of genes more versatile, rapid, and large-scale communications of controlled by the cognate acetaldehyde-inducible pro- communities. moters. Therefore, the sender cells produced a concentric Weber et al. recently established a communication gradient of acetaldehyde that induced the dose-dependent system that utilizes acetaldehyde as signaling molecules gene expression of the receiver cells with the expression [51]. In their study, a bacterial strain (sender) was engi- level defined by the distance between the sender and neered to constitutively express alcohol dehydrogenase receiver cells. (ADH), an enzyme that converts ethanol in the medium In another example, Hasty and colleagues constructed to acetaldehyde. Due to its low boiling point (21°C), ndh-2, a gene encoding NADH dehydrogenase II (mem- acetaldehyde volatized and was broadcast to neighboring brane-bound respiratory enzyme), into an Escherichia coli X Kong et al. Bioresources and Bioprocessing 2014, 1:24 Page 4 of 11 http://www.bioresourcesbioprocessing.com/content/1/1/24 strain to confer the production of hydrogen peroxide (A) (H O ) [83]. H O is a thermodynamically unstable chem- 2 2 2 2 ical compound and is able to enter neighboring cells quickly to alter their redox state and inactivate ArcAB, resulting in the shift of the activity of the corresponding LuxR AHL downstream genes. Through the exploitation of H O ,a 2 2 novel route of airborne signaling molecule was created for fast and large-scale colony coordination. ccdB luxI LuxI Other communication mechanisms LuxR In addition to the common signaling mechanisms discussed above, bacteria also exploit a wide range of alternative Cell death approaches for communications, such as quinolone signal luxR luxI [84], diffusible signal factor [85], cyclic dipeptide [86], con diketopiperazines [86,87], and others [88,89]. One such representative mode of signaling is the use of indole, an (B) aromatic heterocyclic organic compound that is produced AHL by over 85 species of Gram-positive and Gram-negative bacteria and used as an extracellular signal for global LuxR coordination of various bacterial species [90]. Although ccdA LuxI little of those mechanisms have been explored for luxI LuxR synthetic biology applications, the broad spectrum of signaling systems provides a rich reservoir for engineering luxI luxR multicellular functionality. lac/ara Dynamic group behaviors of bacterial communities via engineered communications Cell death ccdB Cellular communications enable the coordination of sin- lac gle cells by sending and sensing the states of individuals. Figure 2 Cellular density control enabled by engineered Inspired by this natural capability of bacteria, synthetic cellular communications. (A) A communication-based gene circuit biologists have developed a set of engineered bacterial that confers the auto-regulation of cellular population density. In this populations with their group behaviors programmed from system, a positive correlation between cell density and AHL concentration designed artificial cell-cell communications. is essential and was created by having the cells constitutively produce LuxI that catalyzes AHL synthesis. At a low density, cells survive and grow normally because the expression of the toxin gene ccdB is not activated Population density control by a low AHL level. In contrast, when cell density achieves a critical level, The first communication-based synthetic circuit was ccdB expression is triggered by accumulated AHL, causing cellular death. built by You et al. in 2004 with the goal of creating a The density-dependent cell death ensures an automatic control of total dynamic, autonomous regulation of the cell density of population density. (B) A synthetic gene circuit conferring an Allee an E. coli population [18]. As illustrated in Figure 2A, effect in an isogenic population. Rather than the toxin CcdB in (A), the antitoxin CcdA was correlated with cellular density via AHL the Lux system from V. fischeri was introduced to con- concentration. At a low cell density, the cells cannot survive because struct cell-cell communication and was coupled to cell of their production of the toxin CcdB. At a high density, the production survival and killing via the CcdA/B toxin system. Here, of the antitoxin CcdA is triggered to neutralize the toxic effects from the LuxI protein catalyzes the synthesis of a small, dif- CcdB, resulting in normal cell growth. fusible AHL signaling molecule, 3OC HSL, which accu- mulates in the extracellular milieu and the intracellular environment as the cell density increases. When cells state. Indeed, a stable cell density was maintained for reach a sufficient density, the AHL binds to LuxR and more than 30 h with the variation within less than 5% in forms the LuxR/AHL complex that activates the expres- the study. This density control circuit laid a foundation sion of the killer protein LacZα-CcdB, leading to cell for using cellular communications to program bacterial death. On the other hand, cell death can cause a reduc- communities, allowing the extension of the control of tion of total population density and hence the level of population dynamics to the engineering of more sophisti- AHL production, which in turn allows the population to cated synthetic ecosystems. recover after killing. The continuous production and deg- In a recent work, Smith et al. utilized the density control radation of AHL make the cell density approach a steady circuit constructed above to create an artificial Allee effect Kong et al. Bioresources and Bioprocessing 2014, 1:24 Page 5 of 11 http://www.bioresourcesbioprocessing.com/content/1/1/24 in E. coli populations [91]. The Allee effect is a biological (A) phenomenon characterized by a correlation between population density and the mean individual fitness of a population [92]. To create such an effect, a synthetic gene circuit was constructed to contain the LuxI/LuxR system and the CcdA/B toxin-antitoxin system (Figure 2B). In LuxR this setting, the expression of LuxR/LuxI and CcdB (killer) is under the control of P promoter, while CcdA lac/ara AiiA luxR (rescue) was regulated by the cell density-dependent P lux luxI con promoter. When IPTG induction is on, the cellular popu- GFP LuxR lation growth rate is negative if the initial cell density is aiiA less than the critical value (C ) at which CcdA expres- crit gfp sion is not activated. However, if the initial cell density is luxI above C , AHL activates the production of LuxR and crit further drives the production of CcdA which rescues the (B) population by inhibiting the toxicity of CcdB. An Allee effect population was thus established to have a negative fitness below a threshold of cell density but a positive LuxR fitness when the density is beyond the threshold. This study provided new implications of engineered cellular luxR AiiA communication for controlling invasive species and the P luxI con spread of infectious diseases. GFP LuxR aiiA Temporal synchronization NDH gfp Complex cellular behaviors, such as biofilm formation and host invasion, often require the temporal coordination and ndh collective action of cellular populations [93,94]. Towards luxI this need, engineered communications offer a powerful solution. In a recent study, Hasty and colleagues reported the de- Figure 3 Synchronization of genetic oscillations by velopment of an artificial gene circuit that synchronizes communication-based circuits. (A) A QS-based gene circuit the oscillation of gene expression in individual cells [13]. that synchronizes the oscillation of gene expression in an isogenic Figure 3A shows their circuit design based on the QS bacterial population. The oscillation is enabled by positive feedback, arising from the self-activation of AHL synthesis, and negative elements of V. fischeri (luxI, luxR)and Bacillus thurigensis feedback, mediated by the AHL-degrading gene aiiA. The coupling (aiiA). The AHL 3OC HSL, synthesized by LuxI, of the two feedback loops results in robust oscillations of gene binds to transcriptional factor LuxR to form a complex expression of an entire bacterial population (thousands of cells). (LuxR-AHL) that activates the expression of luxI,which (B) An advanced gene circuit modified from (A) that enables leads to a positive feedback loop in regulation. At the large-scale synchronization of oscillatory gene expression. In addition to the coupled positive and negative feedback in (A), an additional same time, the LuxR-AHL complex also activates the positive feedback loop is introduced by coupling the production of expression of aiiA, a gene encoding the AHL degradation thermally unstable H O by NADH dehydrogenase (ndh)withAHL 2 2 enzyme, which leads to a negative feedback loop in biosynthesis, leading to global oscillation synchronization of millions regulation. The dual positive and negative feedback of cells. loops drive the sustained oscillation of gene expression of individual cells, and in the meantime, the signaling molecule AHL confers the synchronization of individual Building on their success of the synchronized oscillator, oscillations. Using a custom-tailored microfluidic device, the same group further advanced to create a more sophis- the authors were, for the first time, able to establish ticated genetic network that is capable of synchronizing and tune synchronized oscillations of an entire cellular oscillatory gene expression of populations across multiple population (thousands of cells). Compared with the spatial scales [83]. As illustrated in Figure 3B, the resear- single cell oscillators developed by the same group [11] chers placed a copy of the ndh-2 gene, which encodes and other researchers [1,10], the engineered cellular NADH dehydrogenase II, under the control of an add- communication indeed conferred the synchrony of cellu- itional copy of P promoter (compared with Figure 3A). lux lar gene expression dynamics at a robust and yet tunable The NDH-2 produces a low level of H O that vapors and 2 2 fashion. passes through the walls of the oxygen-permeable LuxI AHL LuxI AHL H O 2 2 Kong et al. Bioresources and Bioprocessing 2014, 1:24 Page 6 of 11 http://www.bioresourcesbioprocessing.com/content/1/1/24 polydimethylsiloxane (PDMS) chips. Driven by the oscilla- organs, or even entire organisms, one critical step is to tion of gene expression mediated by the AHL-based syn- develop an engineering strategy that enables robust spa- chronized oscillation circuit, H O was periodically tiotemporal pattern formation of living cells. Engineered 2 2 produced and exchanged between the cells within individ- cellular communications hold a great promise towards ual chambers. When entering cells, H O changes the this goal, in addition to their roles in conferring temporal 2 2 redox state of the cells and inactivates their lux promoter coordination of cellular behaviors. binding protein ArcAB, causing the global activation of In fact, synthetic biologists have already made several the lux promoter of the cells in different chambers. As a interesting attempts through the exploitation of artificial result, thousands of oscillating colony ‘biopixels’ (appro- communication-based gene circuits. For instance, Sohka ximately 2.5 million cells) were synchronized over et al. constructed a circuit implementing Wolpert's French centimeter-length scales through the use of synergistic flag model [96], enabling the determination of cell fates in intercellular coupling involving both quorum sensing a concentration-dependent manner [25]; Payne et al. cre- within a colony and gas-phase redox signaling between ated a circuit that allows self-organized pattern formation colonies. As a proof-of-concept application, this system without morphogen gradients in bacteria [97]; Basu et al. was further employed to sense arsenic in environments engineered a band detector that allows for differential via differential modulations of the period of the oscillatory response of gene expression according to the local cells that resemble a liquid crystal display (LCD)-like concentration of AHL, creating a bull's eye-like spatio- macroscopic clock. temporal pattern [16]. One elegant example for this line of applications is the Spatial organization programming of bacterial stripe patterns by Liu and co- One of the most fascinating aspects of biological systems workers [49]. As shown in Figure 4A, the gene circuit is their ability to generate complex but highly reproducible consists of two functional parts: density-sensing module organisms through differential spatial patterning of mor- and motility-control module. The density-sensing module phogens across isogenic cells [95]. Towards the ultimate centers on the LuxI/LuxR QS system that enables the syn- goal of biological engineering for creating desired tissues, thesis and excretion of the AHL and the activation of the (A) (B) Red light EnvZ OmpC LuxR AHL P LuxR luxR AHL con cI PCB cph8 LuxR luxI LuxR LuxI con cI LuxI cI Z CheZ pcyA cI ho1 luxI lacZ luxR cheZ luxI P P con ompC lux-lambda lambda con Figure 4 Spatial organization of cellular populations via engineered communication circuits. (A) A genetic circuit that generates periodic stripes in space. The LuxI/LuxR QS system is coupled to cellular motility via the transcriptional repressor gene cI, which is induced by AHL via the promoter P and inhibits the expression of cheZ, one of the essential genes in bacteria motility. At a low cell density, AHL concentration remains low luxI and no CI is produced, leading to a constant production of CheZ and hence a high cell motility. In a high cell density, sufficient accumulation of the AHL induces CI production which in turn suppresses cheZ expression, resulting in a deficiency in cell motility. The density-dependent motility of the population generates periodic stripe patterns in an expanding cell population. (B) A multi-module, communication-based synthetic circuit that allows accurate edge detection. Three functional modules are involved, including a light sensor, a cell communication module, and an X AND (NOT Y) gate. Upon exposure to red light, the light-sensing protein Cph8 induces the expression of cI and luxI: CI represses the expression of lacZ in the same cell regardless of AHL concentration while LuxI triggers the production of AHL that can diffuse to neighboring cells in the dark region to induce the production of LacZ. On the other hand, cells far from the light cannot produce LacZ because no AHL is available to trigger the transcription. As a result, only the cells near the edge of the light-exposed area actively express lacZ, which results in a dark pigment due to the enzymatic cleavage of a substrate in the plate by LacZ. Kong et al. Bioresources and Bioprocessing 2014, 1:24 Page 7 of 11 http://www.bioresourcesbioprocessing.com/content/1/1/24 downstream gene cI when cell density is sufficiently high. Figure 5A shows the design of the ecosystem that The motility-control module is based on the bacterial involves two QS modules, LuxI/LuxR from V. fischeri and motility system that is regulated by the transcription of LasI/LasR from P. aeruginosa, for two-way communi- cheZ. Upon the replacement of the wild-type cheZ cations. The predator cell (top) produces and secretes the with an inducible version (cheZ is under the control of the AHL 3OC HSL that induces the expression of the toxin cI-repressed P promoter), cellular motility becomes gene ccdB in the prey cell (bottom), leading to the death lambda regulated by the expression of cI. With the coupling of the of the prey. In the meantime, the prey produces another two modules, engineered E. coli populations were able AHL molecule, 3OC HSL, which rescues the predator by to form robust but tunable periodic stripes of high and inducing the production of antitoxin CcdA that neutral- low cell densities sequentially and autonomously. These izes the toxin from CcdB. With appropriate modulations results established cellular motility as a simple route to of the system parameters, the researchers were able to create recurrent spatial structures without the need for create a bacterial version of predation with different an extrinsic pacemaker. As a novel mechanism, it offered population dynamics generated, including extinction, an alternative solution for the formation of biological coexistence, and oscillation. Similar to this work, another spatial patterns that is distinct from the well-acknowledged bidirectional intercellular communication network was Turing mechanism [98]. also engineered by Brenner et al. [50], in which the LasI/ In addition to autonomous pattern formation, the LasR and RhlI/RhlR QS systems from P. aeruginosa were QS-based communication mechanism can also be applied adopted to create a two-species microbial consensus to detect complex spatial signals. Tabor et al. recently consortium. In that ecosystem, the gene expression of developed a multi-module gene circuit system for edge any of the two species mutually depends on the presence detection, a signal processing algorithm common in artifi- of the other. cial intelligence and image recognition [99]. As illustrated Beyond predation and consensus, designer cellular in Figure 4B, the biological edge detection algorithm is communications can be used to create a wide spectrum composed of three modules: a dark sensor (NOT light), of inter-species interactions. As revealed by metagenomics cell-cell communication cassette, and an X AND (NOT Y) and 16S pyrosequencing, microbial interactions in nature genetic logic. The darker sensor was engineered based on such as biofilms and the microbiome are extremely com- the light-sensitive protein Cph8, a chimeric sensor kinase. plicated and diverse - for instance, there can be parasitism, With the covalent association of chromophore phycocya- predation, commensalism, mutualism, competition, and nobilin produced from heme via ho1 and pcyA [100,101], amensalism within a single pair of species [102]. As one of Cph8 is able to activate the ompC promoter (P )by the earliest efforts towards the programming of com- ompC transferring a phosphoryl group to the response regulator plicated cellular consortia, Weber and Fussenegger devel- OmpR. However, in the presence of red light, the kinase oped a set of pairwise interactions between E. coli and activity of Cph8 is inhibited, which precludes the tran- Chinese hamster ovary (CHO) cells [51]. scription from P and causes a NOT light trans- As illustrated in Figure 5B, the designs of the ecosystems ompC criptional logic gate. The cell-cell communication was center on an airborne transmission of the transcription sys- implemented through the Lux QS system and was used to temthatallowsone species(E. coli) to convert ethanol into convert light information into spatial distribution of AHL. volatile acetaldehyde and broadcast this airborne signal With the incorporation of the converter cI and the hybrid (boiling point: 21°C) to another species (CHO-K1 cell line) promoter P ,the stateofP is converted via an for the activation of functionally specific, rationally engi- lux-lambda ompC X AND (NOT Y) logical operation into the state of the neered genes. The commensal ecosystem (top) was created promoter P , which is displayed via the production by constructing an E. coli strain capable of converting lux-lambda of LacZ that produces black pigment. Upon the loading of ethanol into acetaldehyde for air broadcast and placing a the programs, a lawn of isogenic E. coli populations was neomycin resistance gene (neo) under the control of an able to sense an image of light, communicate to identify acetaldehyde-induced promoter (P ) in a CHO-K1 cell air the light-dark edges, and visually present the result of the line. In addition, secreted alkaline phosphatase (SEAP) computation. was used as a reporter of the CHO-K1 cells. When cul- tivated proximate to synthetic CHO-K1, the engineered Ecosystem formation E. coli cells confer survival of the mammalian cells while Artificial cellular communications can enable not only keeping their own growth unaffected by the mammalian the coordination of isogenic cell populations but also cells cultured in a separate dish. The amensal ecosystem heterogeneous ecosystems that are composed of multiple (middle) was synthesized by cultivating an acetaldehyde- species. You and co-works recently developed two gene broadcasting E. coli strain in close proximity to a CHO-K1 circuits into a predator-prey ecosystem that consists of cell line that was engineered to have acetaldehyde- two E. coli populations [52]. controlled expression of RipDD, a gene that encodes an Kong et al. Bioresources and Bioprocessing 2014, 1:24 Page 8 of 11 http://www.bioresourcesbioprocessing.com/content/1/1/24 (A) (B) (C) Commensalism Culture well 2 Neomycin Culture O Cell death Initial colonizer cell Predator well 1 AlcR Cell growth + EtOH P NEO air CP25 Disperse lasR rfp A E. coli neo ccdB O SEAP lac/ara-1 P SV40 LtetO-1 seap BdcAE lasI luxR LasR ccdA RFP 50Q luxI LasR LuxR bdcAE50Q LasI LuxR Amensalism lasI Culture well 2 Culture O well 1 3OC HSL 6 3OC HSL O Cell growth 12 + EtOH AlcR Apoptosis E. coli 3OC HSL Disperser cell RipDD Prey 12 Disperse ripDD Cell death P air LasR Hha GFP LasI B 13D6 LuxI LasR Mutualism Culture well 2 Neomycin gfp lasI hha13D6 Culture O luxI lasR ccdB CP25 P T5-lac P P well 1 luxI lac/ara-1 AlcR Cell growth + EtOH air sBLA neo E. coli SEAP NEO SV40 seap Ampicilin Figure 5 Programmed ecosystems developed from designed cellular communications. (A) A synthetic predator-prey ecosystem in E. coli. The predator cell (top) produces the QS signal 3OC HSL that induces the expression of the toxin gene ccdB in the prey cell (bottom) and causes cell death. Meanwhile, the prey produces another QS signal, 3OC HSL, which triggers the expression of ccdA, an antitoxin gene whose expression rescues the predator by neutralizing the toxin CcdB accumulated inside the cell. (B) Synthetic ecosystems with E. coli and Chinese hamster ovary (CHO) cells. The three ecosystems were based on the same foundation - an airborne transmission of transcription system, through which the sender (E. coli) converts ethanol into volatile acetaldehyde and broadcasts it to the receiver (CHO-K1) to alter corresponding gene expression. Top panel: The volatile acetaldehyde produced by E. coli induces antibiotic resistance in CHO-K1 cells, leading to a commensal community. Middle panel: The acetaldehyde from the sender induces the apoptosis of the receiver, creating amensalism between the two species. Bottom panel: E. coli rescues the CHO-K1 cell by triggering antibiotic resistance through volatile acetaldehyde, and in the meantime, the CHO-K1 cell benefits E. coli by degrading ampicillin that is toxic to E. coli, resulting in a mutualistic consortium. (C) An engineered biofilm-forming system that consists of two communicating E. coli species. The disperser cell (bottom) produces AHL (3OC HSL) to trigger the expression of the gene bdcAB50Q in the initial colonizer cell (top), leading to the dispersion of the biofilm formed by the initial colonizer cells. Meanwhile, the biofilm formed by the disperser cells can also be dispersed by inducing the expression of the dispersal protein Hha13D6 with external inducer IPTG. The combination of the two steps allows the replacement and removal of biofilms in a programmed manner. apoptosis-inducing human receptor interacting protein. understanding and programming microbial community As a result, the CHO-K1 cells survive only in the absence patterns that orchestrate the complex coexistence of living of the E. coli cells because, otherwise, they induce the systems. death of the CHO-K1 cells by producing acetaldehyde. In addition to programming planktonic bacterial popu- To create a mutualistic interaction between E. coli and lations, synthetic communication circuits have also been CHO-K1 cells (bottom), the commensal ecosystem devel- exploited in controlling complex communities such as oped earlier (top) was modified to incorporate a mamma- biofilms. Hong et al. recently developed quorum-sensing lian beta-lactamase gene sBLA under the control of the circuits to program the formation and dispersal of acetaldehyde-inducible promoter (P ). Here, sBLA can be artificial E. coli biofilms [103]. As shown in Figure 5C, the air secreted to the extracellular milieu to hydrolyze the circuits have two functional parts with one belonging to bacterial antibiotic ampicillin in the culture medium the initial colonizer cell (top) and the other belonging to to promote the survival of co-cultured E. coli,resulting the disperser cell (bottom). The initial colonizer part in bidirectional benefits between the two cell species. consists of the constitutively expressed repressor gene Following a similar idea, three additional types of eco- lasR and its cognate promoter P that drives the expres- lasI system interactions were created, including parasitism, sion of the biofilm dispersion gene bdcAB50Q;the third party-inducible parasitism, and predator-prey inter- disperser part is composed of the AHL-producing gene action (not shown in Figure 5). This example demon- lasI that is constitutively expressed and another biofilm strated the ability of programming microbial consortia via dispersion gene, hha13D6, controlled by external inducer rational design of cellular interactions by rewiring cellular IPTG. Such a design allows the disperser cell to trigger communication systems, providing novel insights in the expression of the gene bdcAB50Q in the initial SBLA Kong et al. Bioresources and Bioprocessing 2014, 1:24 Page 9 of 11 http://www.bioresourcesbioprocessing.com/content/1/1/24 colonizer cell by producing AHL (3OC HSL), leading to productions [115-117]. There are a variety of research the dispersion and replacement of the biofilm formed by fields where synthetic bacterial consortia have started to the initial colonizer cells. Meanwhile, the circuit in the play an important role: In metabolic engineering, cellular disperser enables the biofilm formed by the dispersers to communication can be used to implement self-regulated be removed with the external signal inducer IPTG. These control between cellular growth and product manufac- types of functional circuits can be powerful in creating turing in bioreactors for autonomous bioproduction. In designer biofilms and enabling precise manipulation of biomedical applications, custom-tailored probiotic bac- community composition in the fields of biorefinery, teria can be introduced into the human body to alter the medicine, and bioproduction. composition and hence the function of the gut microbiota for disease treatment. In areas relating to the environ- Conclusions ment, biofilms and microbial consortia in soil and other With the advances of synthetic biology technologies and a natural settings can be perturbed and even reprogrammed consensus on the need for community-based functionality with engineered microbes for desired purposes. We thus engineering, synthetic microbial consortia have undergone expect that microbial communities programmed via a rapid development in the past few years. This review has engineered cellular communication will become a versa- surveyed recent advances of engineered biological systems tile strategy in addressing both scientific and practical that utilize cell-cell communication to program bacterial challenges in the near future. group behaviors, covering both the basic communication Abbreviations modules and functional gene circuits that confer desired ADH: alcohol dehydrogenase; AHL: acyl-homoserine lactones; AI-2: autoinducer-2; community-based dynamic behaviors. AIP: auto-inducing peptide; CHO cells: Chinese hamster ovary cells; Although there has been significant progress, the en- H O : hydrogen peroxide; Ndh-2: NADH dehydrogenase II; QS: quorum 2 2 sensing; SEAP: secreted alkaline phosphatase. gineering of microbial communities is still in its infancy and is subject to a set of challenges. In fact, almost all Competing interests synthetic circuits to date have involved many rounds of The authors declare that they have no competing interests. trial and error before achieving the desired functionality. Difficulties in the efficient construction of engineered Authors' contributions circuits often stem from a lack of biological knowledge. QH and TL conceived the study and designed the project. WT, VC, and TL drafted the manuscript. CL analyzed the data. All authors read and approved Specifically, to facilitate gene circuit engineering, it is the final manuscript. needed to have a deep understanding of stochasticity in gene expression [104-106], the inherent interplay Acknowledgements between a synthetic circuit and the host organism [1], We thank Andrew Blanchard for commenting and editing the manuscript. This work was supported by the American Heart Association (Grant No. and issues related to multicellular physiology and metab- 12SDG12090025), the Network for Computational Nanotechnology at UIUC olism [107]. Another big challenge arises from the tech- sponsored by National Science Foundation (Grant No. 1227034), and the nical side of synthetic biology, which includes the lack of UIUC Research Board. powerful rational design platforms, limited availability of Author details parts and modules, efficient systematic optimization strat- Department of Bioengineering, University of Illinois at Urbana-Champaign, egies and toolkits, and high-throughput assays for circuit 1304 W Springfield Avenue, Urbana, IL 61801, USA. Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W Gregory Drive, validation. Addressing the above challenges will foster our Urbana, IL 61801, USA. State Key Laboratory of Bioreactor Engineering, East engineering capability and help to achieve the ultimate China University of Science and Technology, 130 Meilong Road, Shanghai goal of efficient and reliable development of synthetic 200237, People’s Republic of China. 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"Bioresources and Bioprocessing" – Springer Journals

Published: Dec 1, 2014

Keywords: Biochemical Engineering; Environmental Engineering/Biotechnology; Industrial and Production Engineering

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