Abstract
Bacterial cellulose is a strong and flexible biomaterial produced at high yields by Acetobacter species and has applications in healthcare, biotechnology and electronics. Naturally, bacterial cellulose grows as a large unstructured polymer network around the bacteria that produce it, and tools to enable these bacteria to respond to different locations are required to grow more complex structured materials. Here, we introduce engineered cell-to-cell communication into a bacterial cellulose-producing strain of Komagataeibacter rhaeticus to enable different cells to detect their proximity within growing material and trigger differential gene expression in response. Using synthetic biology tools, we engineer Sender and Receiver strains of K. rhaeticus to produce and respond to the diffusible signalling molecule, acyl-homoserine lactone (AHL). We demonstrate that communication can occur both within and between growing pellicles and use this in a boundary detection experiment, where spliced and joined pellicles sense and reveal their original boundary. This work sets the basis for synthetic cell-to-cell communication within bacterial cellulose and is an important step forward for pattern formation within engineered living materials.
Introduction
Cellulose is a simple, yet versatile glucose polymer from which biology weaves a broad array of materials. It is one of nature’s most abundant polymers, and can form the basis of materials that are light and elastic, such as a loofah, or structures that are strong and stiff like bamboo (Youssefian et al., 2015; Chen et al., 2017). Cellulose is produced most abundantly by plants, however, a number of bacterial species naturally overproduce cellulose as part of their biofilm matrix, making a structure known as a pellicle (Römling et al., 2015). Bacterial cellulose differentiates itself from plant cellulose in that it is typically produced as ultrapure nanocellulose fibres, free from the contaminating polymers like pectin and lignin that are co-produced by plant cells. Bacterial cellulose (BC) is well known for being strong and flexible, with a single nanofiber having the tensile strength of ~1 GPa and a Young’s modulus of 114 GPa; whilst also being highly hydrophilic, with water making ~90% of its weight in its wet state (Lee et al., 2014). These material properties have seen commercial uptake of bacterial cellulose for biomedical and cosmetic applications, such as for protective bandaging, while also offering potential as a material used in electronics, e.g. as a battery separator or as a matrix ingredient in organic light-emitting diode (OLED) displays (Lee et al., 2014; Jang et al., 2017). Some BC-producing bacteria have also been demonstrated to be amenable to genetic engineering (Florea et al., 2016), offering the possibility of using cellulose-producing bacteria as a means to produce genetically-defined materials, where the material properties and functions can be programmed by engineering at the DNA level using the modern tools of synthetic biology (Cameron et al., 2014). Programming material properties and features into biomaterials using such tools is part of the emerging new field of engineered living materials (ELMs) (Nguyen et al., 2018).
In most natural systems that produce biomaterials, communication between material-producing cells is a critical component of producing materials with structures on the micro and macro scale. ELMs research can theoretically recapitulate this in material-producing bacteria by leveraging the significant past work done to engineer Escherichia coli for synthetic pattern formation (Scholes et al., 2017). Such engineered pattern formation work typically exploits quorum-sensing systems from various bacteria, which are a natural method of bacterial cell-to-cell communication that can be reprogrammed (Scholes et al., 2017). The Lux quorum-sensing system from Vibrio fischeri is particularly well-used in this context, utilising the signalling molecule 3OC6-HSL – often referred to generally as acyl-homoserine lactone (AHL) (Churchill et al., 2011). AHL is produced by the enzyme LuxI, this diffusible molecule can then bind and activate the transcription factor LuxR, which in activates the pLux promoter to drive expression of a downstream gene (Waters et al., 2005). The diffusible and inducible nature of AHLs can be exploited to create a morphogen gradient - the basis for many models of patterning, such as the French Flag Model (Wolpert, 1969) and Turing patterns (Turing, 1952) (Fig. 1A). In synthetic pattern formation research, the Lux system has been used to produce programmable bullseye patterns on dishes of engineered E. coli cells that produce AHL (‘Senders’), while being surrounded by a lawn of cells that express fluorescent proteins in response to different levels of AHL (‘Receivers’) (Basu et al., 2005). Since then, more complex systems have been engineered using AHL signalling in E. coli, including tuneable stripe patterns with motile cells (Liu et al., 2011), hierarchical patterning with multiple AHLs (Boehm et al., 2018) and the recent creation of stochastic Turing patterns (Karig et al., 2018).
Previously we isolated a genetically-tractable BC-producing Komagataeibacter rhaeticus strain and developed a basic kit of synthetic biology parts and tools to enable its genetic engineering (Florea et al., 2016). Here, we extend our initial K. rhaeticus toolkit to now add engineered cell-to-cell communication to enable downstream work in patterning structural and functional properties into BC-based ELMs. By modifying the AHL-based systems developed previously for E. coli, we introduce and characterise cell-to-cell communication between engineered sender and receiver K. rhaeticus cells and demonstrate this system to be active within pellicles and able to define the boundaries between pellicle regions.
Results
Construction of Sender and Receiver plasmids
To establish synthetic cell-to-cell communication in K. rhaeticus, we designed two separate strains, a Sender strain that produces the diffusible AHL molecule N-(3-oxohexanoyl) homoserine lactone (3OC6-HSL), and a Receiver strain that can sense AHL and respond by expressing an easy-to-measure reporter gene (Fig. 1B). The sender plasmid, pSender, was constructed by placing the AHL-synthesis gene luxI downstream of the synthetic constitutive promoter J23104 (Fig. 1C). The receiver plasmid, pReceiver, used promoter J23104 for expression of the gene luxR and the LuxR-regulated promoter, pLux for expression of the monomeric red fluorescent protein (mRFP) gene (Fig. 1C). The J23104 constitutive promoter was used in both instances as it was previously shown to have high activity in K. rhaeticus (Florea et al., 2016).
AHL production by Sender strains
We first determined whether our engineered Sender K. rhaeticus cells produced and released meaningful quantities of AHL to trigger cell-to-cell communication. To measure effective AHL production levels we used E. coli transformed with pReceiver as an assay system, as E. coli with similar receiver plasmids give a well-characterised increase in reporter gene expression in response to increasing amounts of AHL (Canton et al., 2008). Standard concentrations of chemically-produced AHL were added to the growth media of E. coli Receiver to produce a dose response curve where relative mRFP production levels in E. coli correspond to known levels of AHL (Fig. 2A). We next grew K. rhaeticus Sender cells, and a control K. rhaeticus containing only an empty plasmid (pEmpty), collected their spent growth media and used the E. coli Receiver cells to estimate the AHL concentrations produced. For the K. rhaeticus Sender cells, spent growth media induced mRFP production in E. coli that matched nanomolar levels of synthetic AHL on the dose response curve (Fig. 2A.). Interestingly, detectable levels of E. coli mRFP production were also observed when spent growth media from the K. rhaeticus pEmpty control cells were used, but not when unused HS media was added.
Next, the engineered K. rhaeticus Receiver cells were assessed for their ability to respond to AHL and express the mRFP reporter gene. Spent media from K. rhaeticus Sender was applied to K. rhaeticus Receiver liquid cultures (and control cells) and mRFP production levels determined. K. rhaeticus Receiver strains produced significant levels of mRFP in response to K. rhaeticus Sender spent media, giving approximately 10-fold more red fluorescence than background levels (by comparing pEmpty control strains against K. rhaeticus Receiver). The engineered pReceiver plasmids, using the J23104 promoter for luxR expression, also showed improvement in the sensitivity of the AHL-response system in K. rhaeticus, producing more mRFP from the same input when compared to the previously described pLux01 construct (Florea et al., 2016) that uses a weaker promoter for luxR expression (Fig. 2B).
Cell-to-cell communication between Sender and Receiver strains
Having determined that Sender and Receiver K. rhaeticus could produce and respond to appropriate levels of AHL in isolation, we next examined direct cell-to-cell communication between mixed cultures of the two strains. To investigate interactions at the single cell level, we first examined our co-cultures via fluorescence microscopy. This was enabled using a microfluidic plate set-up that provides continual slow perfusion of new growth media. The set-up allowed K. rhaeticus to grow in a single focal plane and yielded single-cell resolution images of the cultures. Sender and Receiver K. rhaeticus strains were mixed at low density in a 1:1 ratio and incubated at 30 °C in the microfluidic plate system for 48 hours. After this, images of the co-culture were taken and these revealed strong expression of mRFP in clusters of cells surrounded by those not showing any red signal (Fig. 3A). These expressing clusters are presumably Receiver cells that have grown from an ancestor while surrounded by the non-fluorescent Sender cells. To verify this, we imaged Receiver cells grown mixed with pEmpty control cells and exposed them to 100 nM of synthetic AHL and saw the same pattern. As a further control, cells of only the K. rhaeticus Receiver strain were also grown in the set-up and showed only very low-level background red fluorescence (Fig. 3A), confirming that only engineered expression of AHL from Sender strains can trigger significant expression of the reporter gene in Receiver K. rhaeticus.
The results so far showed that our engineered K. rhaeticus behave similarly to E. coli engineered with similar constructs in past work, however, unlike E. coli, K. rhaeticus, naturally produces and grows within a macro-scale material structure. Whilst giving many benefits in terms of downstream applications, the growth of the bacteria in a pellicle may alter the way that gene circuits operate, for example by having the cells switch to stationary phase or making cell conditions anaerobic and thus changing the cell state. Therefore, to determine if the engineered communication system can function in this environment, we next switched to pellicle growth experiments using co-cultures of our Sender and Receiver strains. A 50:50 inoculation ratio of Sender and Receiver K. rhaeticus was set-up and grown alongside equivalent growth of the same control combinations used in the microscopy experiment (Fig. 3B). After 4 days this gave pellicles with the thickness of approximately 5 mm, and when scanned using a fluorescence imager, significant mRFP expression was observed across the pellicle in the Sender+Receiver combination. No fluorescence was observed with just Receiver cells or in the control pellicle where pEmpty was present in cells mixed with the K. rhaeticus Receiver. Fluorescence was observed when synthetic AHL was added exogenously at 100 nM AHL to this control pellicle (Fig. 3B), although the signal was not as strong as for the engineered co-culture, presumably because local AHL concentrations are higher and continually produced when it is being produced within the material.
Cell-to-cell communication between Sender and Receiver pellicles
Past work in E. coli synthetic biology has established that there are many downstream applications once cells within a co-culture can be engineered to communicate (Basu et al., 2005; Boehm et al., 2018; Karig et al., 2018). Given the unique situation of having now established such a system in a material-producing microbe, we decided to investigate if it could be used to mediate communication between whole pieces of grown materials. In engineered living materials this could be used, for example, to determine the edges of grown areas and trigger gene expression at section boundaries. To demonstrate this, Sender and Receiver K. rhaeticus pellicles were grown separately, before placed together in a plate with HS media for 24 hours. Control experiments were also done in parallel, where pellicles from cells containing pEmpty were placed with Receiver pellicles and co-incubated for 24 hours with and without exogenous synthetic AHL. As before pellicles were imaged using a fluorescent scanner, and the images of the pellicle pairs show that the Receiver pellicle only activates mRFP expression when co-incubated with the Sender pellicle or when exogenous AHL was added (Fig. 4A). The signal is strongest at the pellicle edges, representing the areas that see new growth of cells and have the maximum exposure to diffusing AHL coming from the Sender pellicles.
Having verified that the Sender pellicle can produce enough AHL to diffuse out and induce gene expression in Receiver pellicles in just 24 hours, we next moved to boundary detection. In this case, cut Sender and Receiver pellicles were placed adjacent to one another and left to continue to grow in media so that they fuse together to make a larger piece. This technique of fusing pellicles via growth can aid in growing large material products. After only 24 hours incubation of closely adjacent pellicles, fusion had occurred along most of the boundary, now creating a single pellicle. This was imaged as before and revealed a clear line of strong mRFP expression along the edge of the previous Receiver pellicle. This line of expression reveals where the border of the fusion has occurred and in future engineered strains could be the location of the expression of other proteins, such as those that provide stronger material bonding (Fig. 4B).
Discussion
Here we constructed a unidirectional cell-to-cell communication system in the cellulose producing bacteria K. rhaeticus using a system of two separate engineered strains: Sender and Receiver. We utilised the Lux quorum sensing system, commonly used in artificial pattern formation experiments, and showed it to be functional in K. rhaeticus, even within growing bacterial cellulose pellicles. Previous research on synthetic patterning of bacterial cells has entirely focused on patterns formed by colonies on agar surfaces, cells in microfluidic chambers or multi-well plates, whereas here we demonstrate this system working as the engineered cells grow in and around their own macro-material structure.
The work presented here offers a route towards the development of more intricate gene regulation and signalling circuits within growing pellicles made by K. rhaeticus, and will be enabled in the future by the addition of further signalling and regulatory components. Increasing the complexity of cell-to-cell communication, e.g. by bringing in a second diffusible signalling molecule, would allow the realisation of more elaborate patterns, such as Turing patterns, and also enable bidirectional communication between engineered cells. Interestingly, our initial characterisation with E. coli Receiver strains showed that un-engineered K. rhaeticus produces other molecules that partially trigger the Lux system response in E. coli (Fig. 2A). This hints at an a unknown native quorum system in K. rhaeticus which could be further investigated and potentially exploited as a second communication route. While this triggered some expression in Receiver E. coli, it notably did not cause a response in Receiver K. rhaeticus (Fig. 3), suggesting that it is not the exact same AHL molecule. A genomic search of K. rhaeticus only identifies an orphaned luxR homologue, and no obvious AHL synthases (Florea et al., 2016), however, recent work has shown that a closely-related Acetobacter strain does produce quorum molecules (Liu et al., 2018).
A direct potential utility of the boundary detection shown here (Fig. 4), could be to create a self-repairing material that re-joins cuts with stronger attachment than just natural pellicle fusion. This could be achieved by replacing the mRFP gene in the pReceiver construct by genes that express proteins that promote stronger attachment, e.g. by the surface display of adhesive catecholamines (Park et al., 2014) or via production of bacterial adhesive fibres such as Curli (Nguyen et al., 2014). Our work could also be further expanded by combining our signalling with genetic logic gates that link cell-to-cell communication with decision making and the response to combinations of external and internal stimuli (Brophy et al., 2014; Scott et al., 2016; Liu et al., 2018). Overall, we believe that a living bacterial cellulose material capable of programmable cell-to-cell communication could be adapted for a variety of purposes, and further engineered to create patterned materials with unique macro structure-driven material properties and novel functions.
Experimental Procedures
Strains, plasmids and culturing conditions
E. coli was cultured in at 37°C shaking at 250 rpm in Lysogeny broth (LB) (10 g/l Tryptone, 5 g/l Yeast Extract and 5 g/l NaCl) or statically at 37°C in LB agar with 34μg/ml chloramphenicol when appropriate. Cultures of K. rhaeticus iGEM were grown at 30°C in Hestrin – Schramm media (HS) (20g/l glucose, 10g/l yeast extract, 10g/l peptone, 2.7g/l Na2HPO4 and 1.3g/l citric acid, pH 5.6-5.8) or on HS agar plates (1,5% agar) and supplemented with 2% cellulase, when appropriate 34 μg/ml chloramphenicol was added. Electroporation of K. rhaeticus strains were performed as detailed previously (Florea et al., 2016).
For K. rhaeticus pellicle growth, parental pellicles were first generated by inoculating strains from glycerol stocks in 5 ml HS media and incubated at 30°C for 7 days. In order to maintain plasmids 340 ng/μl chloramphenicol was added. Liquid beneath the parent pellicle was then used, in 1:50 ratio, to inoculate parallel experimental pellicles. Experimental pellicles were harvested after 3-4 days of growth at 30°C. To generate mixed strain pellicles, the media from beneath each parent pellicle was removed and mixed at a 50:50 ratio, this mix was then inculcated into fresh HS media and placed in a deep-well 24 well plate (Axygen).
Plasmids used in this study are listed in Table 1. The vector backbones were based on previous BioBrick compatible vectors (for detailed cloning description see (Florea et al., 2016)) and J23104 promoter was adapted using Phusion polymerase (NEB) and PCR mutagenesis (primers listed in Table 2).
Spent media assays
Spent media was made by growing K. rhaeticus Sender strain to late exponential/early stationary phase, cells were then pelleted, and supernatant filtered before used to preform assays. Spent media or synthetic AHL (product K3255, Sigma Aldrich) was added to exponentially growing cultures and fluorescence measured after an hour. mRFP production was calculated as difference in fluorescence (measured at ex., 590 nm; em., 645 nm) between successive time points normalized by OD600. Multiple assays were performed in 96 well plate format in the Synergy HT Microplate Reader (BioTek) each with triplicate technical replicates. In order to estimate AHL concentration E. coli strain were transformed with relevant vectors, and grown in a 96 well format. Before adding to E. coli cultures spent HS-media was buffered to pH 7 using Tris pH 8.0, this was required as HS media has a low pH that prevent E. coli growth if added directly. K. rhaeticus strains were grown in 25 ml tubes, with vigorous shaking and cellulase, after addition of spent media samples were plated into 96 well plates and fluorescent measurements taken at ex., 590 nm; em., 645 nm within the hour. Cellulose production encouraged clumping in liquid K. rhaeticus cultures, making precise OD measurements for growing liquid cultures challenging, even with high cellulase treatment. Therefore, when adding spent media to K. rhaeticus, the mRFP response was measured within the first doubling time before clumping caused an effect.
Fluorescence imaging
Fluorescence images of pellicles were taken with a Fujifilm FLA-5000 Fluorescent Image Analyser, with excitation at 532 nm, 600 v and Cy5 filter, whilst Pseudo-brightfeild images were taken with excitation at 473 nm, 400 v and FITC filter. Fluorescence microscopy images were taken on a Nikon Eclipse Ti inverted microscope using a Rolera EM-C2 camera. K. rhaeticus strains were grown within a ONIX microfluidics plate (B04A) (Merck) with flowing HS media at 1 psi and incubated at 30°C. brightfield and mRFP fluorescence Images were taken after 48 hours with a 60x oil emulsion objective, Exposure time 1.25 seconds and EM gain of 675. Corresponding brightfield images were contrast enhanced with a phase filter (Ph3). Image processing and compositing was conducted in FIJI (Schindelin et al., 2012).
Pellicle-to-pellicles assays
The separate pellicle pairs were placed within the same petri dish with 5 ml HS media and incubated at 30°C with gentle shaking of 60 RPM. After 24 hours the pellicle pairs were removed, and fluorescence imagery taken. The Boundary detection assay used a larger pellicle, grown for 2 days in a 10×10 cm square petri dish with 50 ml HS media. To produce a uniform pellicle at this larger surface area, a 1:10 ration of the liquid from under the first batch of pellicles was used as inoculum. Sender and receiver pellicles were removed from cultures, excess media was removed, and the pellicles were sliced in half along the diagonal. To facilitate diffusion yet prevent sinking, the two pellicles were placed in proximity on soft 0.6% HS agar and incubated at 30°C for 24 hours before fluorescence imagery was taken.
Author Contributions
V.J.G., K.T.W. and T.E. conceived and designed the experiments, K.T.W., A.D. and A.G. performed the experiments, K.T.W., V.J.G. and A.D. analysed the experimental data, V.J.G., K.T.W. and T.E. prepared the manuscript.
Conflict of Interest
None declared
Acknowledgements
The authors wish to thank Dr Benjamin Reeve and Dr Carlos Bricio-Garberi for initial help with this project and acknowledge the UK Engineering and Physcial Sciences Research Council (EPSRC) for the funding of this work.