Hydrogel-based Bio-nanomachine Transmitters for Bacterial Molecular Communications
Daniel P. Martins, Huong Q.-O'Reilly, Lee Coffey, Paul D. Cotter, Sasitharan Balasubramaniam
HHydrogel-based Bio-nanomachine Transmitters for BacterialMolecular Communications
Daniel P. Martins
VistaMilk SFI Research CentreTelecommunications Software andSystems GroupWaterford, [email protected]
Huong Q.-O’Reilly
Telecommunications Software andSystems GroupPharmaceutical and MolecularBiology Research CentreWaterford, [email protected]
Lee Coffey
VistaMilk SFI Research CentrePharmaceutical and MolecularBiology Research CentreWaterford, [email protected]
Paul D. Cotter
VistaMilk SFI Research CentreTeagasc Food ResearchFermoy, [email protected]
Sasitharan Balasubramaniam
VistaMilk SFI Research CentreTelecommunications Software andSystems GroupWaterford, [email protected]
ABSTRACT
Bacterial quorum sensing can be engineered with a view to thedesign of biotechnological applications based on their intrinsic roleas a means of communication. We propose the creation of a posi-tive feedback loop that will promote the emission of a superfoldedgreen fluorescence protein from a bacterial population that will flowthrough hydrogel, which is used to encapsulate the cells. Theseengineered cells are heretofore referred to as bio-nanomachinetransmitters and we show that for lower values of diffusion coef-ficient, a higher molecular output signal power can be produced,which supports the use of engineered bacteria contained withinhydrogels for molecular communications systems. In addition, ourwet lab results show the propagation of the molecular output signal,proving the feasibility of engineering a positive feedback loop to cre-ate a bio-nanomachine transmitter that can be used for biosensingapplications.
KEYWORDS nanocommunications, engineered bacteria, bio-nanomachines
Bacteria utilise signalling mechanisms (i.e., quorum sensing) todrive collective behaviours within homogeneous and heteroge-neous microbial populations. These biological communicationssystems have been investigated for the past 50 years, and recentlythey became the focus of biotechnological solutions design for bio-fabrication and biosensing [10, 13, 16, 18, 26]. For example, softmatter (i.e. gelatin) was assembled using enzymes emitted by bac-terial cells, and
Escherichia coli bacteria were engineered with asignalling system used by
Vibrio cholerae for a timely detection ofcholerae infection [10, 16]. These and other examples highlight therange of applications that can be designed and built based on theengineering of the bacterial signalling mechanisms. Bacteria signalling has also been investigated using communi-cations theory concepts, in a paradigm named as Molecular Com-munications [1, 2, 6–9, 31]. From this perspective, the bacteria areconsidered as biological bio-nanomachines that are engineered toprocess, emit and detect specific molecules, acting as transceiversfound in conventional communication systems [1, 2, 6, 9]. For in-stance, bacterial quorum sensing has been used to create logiccircuits, modulators and network links between bacterial nodes[7, 8, 20, 31]. Here our focus is on the processing and emission ofmolecular signals as we propose the design of a bio-nanomachinetransmitter that can enable the development of safer intrabodymolecular communications systems in humans and animals. There-fore, we investigate a physical model of a bio-nanomachine trans-mitter embedded in a hydrogel bubble, which will protect the hostenvironment from mixing with engineered cells, and analyse itssuitability for molecular communication systems.This work is inspired by our previous work where we proposedthe production and emission of molecular signals to attract bac-teria to a specific location [19]. In this paper, we focus our inves-tigation on the production and emission of a specific molecularsignal through the hydrogels. A visual representation of the pro-posed work can be seen in Figure 1, where a bacterial populationis placed in a plate with nutrients to grow and produce the de-sired molecular output signal, a fluorescence protein (superfoldergreen flourescence protein–sfGFP). The performance of such a bio-nanomachine transmitter is evaluated with respect to the molecularsignal throughput that is able to reach the border of the hydrogelbubble, and we also study the bacterial growth and nutrient con-sumption associated with the emission of molecules. The contribu-tions of this paper include: • Bio-nanomachine transmitter physical model:
We in-vestigate the molecular signals, the processes related withthe emission of molecular signals, its throughput and signalpower depending on the viscosity of the hydrogel bubbleand internal chemical reactions. a r X i v : . [ q - b i o . CB ] S e p HydrogelAutoinducer BacterialPopulation xhAftersome time
DirectEffect sfGFP
Figure 1: The bio-nanomachine transmitter are embeddedin hydrogel to protect them. The cells are engineered todirectly exchange signals and produce sfGFP for minutesor hours depending on the signalling system used and thequantity of the cells. • Proof-of-concept design:
A wet lab experiment is devisedto demonstrate the emission of a molecular signal, sfGFP,from the bacterial population through the hydrogel.This paper is organised as follows. In Section 2, we characteriseand review the state-of-the-art of biological transmitters. The com-munications and biological models are introduced in Section 3.Section 4 presents the numerical analysis of the molecular emis-sion and throughput. A proof-of-concept design and analysis arepresented in Section 5. Lastly, Section 6 presents our conclusions.
Bacteria produces and consumes different molecular signals throughchemical reactions to collectively establish, protect and maintain acommunity, either single- or multi-species. The internal machinerythat enable these processes can be engineered to produce specificmolecules that will be emitted and propagated through the chosenmedium towards other bacterial populations [22, 28]. To coordi-nate these processes, bacteria often utilises an internal signallingmechanism know as quorum sensing, which is an exchange ofmolecular signals that drive bacteria towards the execution of col-lective behaviours, such as virulence factors production and biofilmformation [4, 19, 21]. The engineering of this process is the targetfor the design and construction of a bio-nanomachine transmitter.Bacterial populations grow and produce quorum sensing mole-cules by consuming the nutrients available in its vicinity. Therefore,a faster nutrient consumption will result in a faster populationgrowth and higher production of quorum sensing molecules. How-ever, this can affect the sustainability of the bacterial population dueto the depletion of its energy source [11, 19, 32]. To avoid such sit-uations, bacterial populations coordinate their collective behaviourdepending on this resource. This coordination is described througha set of chemical reactions that involves strain-specific quorumsensing molecules and receptors, such as LuxI and LuxR (luciferase
LuxI LuxR sfGFPPositive FeedbackOscilator
LuxI LuxRTargetgenes [ sfGFP ] [ sfGFP' ]LuxR HydrogelEdgeMembrane
BacterialPopulation Hydrogel[ sfGFP ] [ sfGFP' ] Transmitter Molecular Channel
Figure 2: Representation of the molecular communicationssystem used to model the emission of molecular signals bythe bacterial population. Adapted from [4] inducer and regulator, respectively) [4]. By engineering this ma-chinery, we are able to create a positive feedback loop using thebacteria quorum sensing process to emit a molecular output signal,sfGFP, and the bacterial population will act as a bio-nanomachinetransmitter. Figure 2 shows a representation of this process, wherebacteria produces LuxI and detects its concentration, using LuxR,creating the positive feedback loop. After being produced throughthis process, the molecular output signal, sfGFP, is then emittedfrom the bacterial population towards the membrane edge of thehydrogel bubble.The engineering of quorum sensing systems through feedbackloops have been proposed before. In [30], a negative feedback loopshave been designed to control the quorum sensing dynamics of
Vibrio harveyi bacteria. Moreover, a positive feedback loop wasengineered to built a whole cell biosensor to detect mercury levels[5]. Here, we propose the design of a positive feedback loop tocharacterise a bacterial population as a bio-nanomachine trans-mitter. One important aspect of our design is the encapsulation ofthe bacterial population in a hydrogel bubble, which is a mediumwith a higher viscosity value and have been previously applied toprotect bacterial populations [14, 27]. Hydrogels are polymers withinfiltrated water that can be applied to encapsulate living cells andhave permeability for a wide range of molecules and facilitate thedesign of contained quorum sensing applications [14, 27]. For oursystem, we propose the use of hydrogel to create an environmentwhere the bacterial population undergoes controlled growth andemits molecular signals, and, at the same time, they are protectedagainst external attacks.
As presented in Section 2, bacteria produce molecular signals by aseries of chemical reactions using their internal machinery, whichcan be engineered for that purpose. In this paper, we engineer the
E.coli population internal machinery using a positive feedback loop to exploit their molecular communications and produce the desiredmolecular output signal, the fluorescence protein sfGFP. Here wedescribe the positive feedback loop using a set of chemical reac-tions (1)-(7) and the propagation of the molecular output signal bysolving the two-dimensional Fick’s law of diffusion to characterisethis molecular communications system. We describe the positivefeedback loop using LuxI and LuxR, represented in Figure 2, asfollows [23] d [ A ] dt = c A + k A [ C ] K A + [ C ] − k [ A ] − k [ R ][ A ] + k [ RA ] (1) d [ R ] dt = c R + k R [ C ] K R + [ C ] − k [ A ] − k [ R ][ A ] + k [ RA ] (2) d [ RA ] dt = k [ R ][ A ] − k [ RA ] − k [ RA ] + k [ C ] (3) d [ C ] dt = k [ RA ] + k [ C ] (4)where [ A ] , [ R ] , [ RA ] , [ C ] are the AHL , LuxR , LuxR – AHL complexand dimerized complex concentration, respectively; c A and c R arethe transcription basal levels for AHL and
LuxR , respectively; k A and k R are the transcription rates; K A and K R are the degradation rates; k , k , k , k , k , k are the translation rates. The rate of molecularsignal production, [ s f GFP ] , by the bacterial population that resultsfrom the quorum sensing process is represented as d [ s f GFP ] dt = k sf GF P [ C ][ C ] + K C . (5)In addition to the production of the sfGFP, the bacterial populationwill also consume nutrients to grow, and the rate of this process isrepresented by d [ GC ] dt = (cid:18) µ [ N ] K N + [ N ] − m (cid:19) [ GC ] . (6)Then, the nutrient consumption rate can be evaluated as follows d [ N ] dt = − U G (cid:18) µ [ N ] K N + [ N ] (cid:19) [ GC ] − U AH L d [ A ] dt − U LuxR d [ R ] dt − U sf GF P d [ s f GFP ] dt , (7)where U G , U AHl , U LuxR , and U sf GF P are utility parameters thatrepresent the nutrient cost of the population growth, autoinducer,receptor and molecular output signal production, respectively. Afterreaching a high concentration, the molecular output signal [ s f GFP ] propagates to the membrane edge of the hydrogel, from where itcan freely diffuse to other engineered or natural bacterial cells. Thepropagation through the hydrogel can be modelled as [17]. [ s f GFP ′ ] = [ s f GFP ]√ πD h t h e − x h D h t h , (8)where, D h , t h , and x h are the diffusion coefficient, the durationand the distance travelled by the molecular signals [ s f GFP ] in thehydrogel h channel, respectively. Time (hours) P r o t e i n C o n ce n t r a t i o n ( m o l/ L ) [sfGFP] =0.000001[sfGFP] =0.00001[sfGFP] =0.0001 [sfGFP] =0.001[sfGFP] =0.01 Figure 3: Evaluation of the molecular output signal through-put for a range of initial concentration values of [ s f GFP ] We evaluate the performance of this communications system usingtwo metrics: the sfGFP throughput from the bacterial populationand the signal power that reaches the hydrogel edge membrane. Thethroughput is evaluated directly from (6), while the signal power isevaluated using (8) and converted into decibel. These metrics areevaluated using the values presented in Table 1.We evaluate the molecular output signal throughput (see Figure3) for 72 hours (this is sufficient time to produce detectable levelsof [ s f GFP ] ), bacterial population of 10 ,
000 cells, a diffusion coeffi-cient of 2 × − (similar value to the model investigated in [12]),and a range of initial concentrations for the [ s f GFP ] from 10 − to10 − mol/L. It can be noted for this scenario, that for the first 10hours there is a steep increase in the production of the molecularoutput signal and a flattening in the throughput after 20 hours formost of the curves. The exception is for the 10 − curve, where thissaturation process only occurs after 50 hours as the positive feed-back loop requires more time to balance all the chemical reactionsrelated to this process due to the high initial value of [ s f GFP ] .We modified the scenario for the throughput analysis, to inves-tigate the signal power that reached the edge membrane of thehydrogel. In this case, we considered the hydrogel as a bubble withdiameter of 7 mm, an initial concentration for the molecular out-put signal [ s f GFP ] of 10 − mol/L; a range of diffusion coefficientvalues from 1 × − cm / s to 2 × − cm / s, and evaluated using(8), where we then converted into db (assuming a 1:1 ratio betweenmol/L and watts), at 24, 48 and 72 hours. In Figure 4 we can see thatthe signal power is higher for lower diffusion coefficients (higherviscosity), and in particular for hydrogels compared to media withlower viscosity, such as water. Therefore, this result infers that thehydrogels or other higher viscosity material can be used to pos-sibly direct molecular signals produced by the bio-nanomachinetransmitters, while the lower viscosity media can be applied foromnidirectional propagation of molecular signals. Diffusion Coefficient (cm /s)
1e 7 S i g n a l P o w er ( d B )
24 hours 48 hours 72 hours
Diffusion in waterHigher viscosityLower viscosityDiffusion in hydrogel
Figure 4: Evaluation of the molecular output signal powerthat reaches the hydrogel edge membrane for low and highviscosity medium.Table 1: Parameters used to evaluate Equations (1)-(8)
Variable Value Unit c A . × − mmol/L c R . × − mmol/L k A × − d − k R × − d − k × − d − k . − k . − k × − d − k . − k . − k sf GF P × − d − K A × − gm − K R × − gm − K C − K N − µ × − gm − m × − gm − U AH L × − – U G U LuxR × − – U sf GF P × − – D h from 1 × − to 9 × − cm / s t h
72 hours x h × − m We prepared wetlab experiments to observe the operation of thepositive feedback oscillator in terms of the amount of molecularoutput signal produced [ s f GFP ] . Bacteria used in this experimentwas E.coli
DH5 α pTD103luxI_sfGFP (abbreviated as E. coli pTD103). pLuxIsfGFPT1 T1T0SpeI (4422)AvrI (3607) AatII (5494) LuxRpLuxIcolE1 pTD103LuxI sfGFP
KanLuxI
LuxI sfGFP
LuxR
Figure 5: Plasmid structure used in this study to producesfGFP due to the increase in production of the molecular in-put signal LuxI. Adapted from [25].
We purchased Luria-Bertani (LB) broth from Fisher Bioreagents ™) ,Agarose from Promega ™ , and Calcium chloride hexahydrate fromMerck ™ . Other reagents and antibiotics required for this exper-iment were purchased from Sigma-Aldrich ™ . Kanamycin (Kan)and Chloramphenicol (Cam) were sterile and utilised at concen-trations of 50 µ g/ml and 34 µ g/ml, respectively. The plasmid usedin this experiment, pTD103luxI_sfGFP, was a gift from Jeff Hasty(Addgene plasmid E. coli pTD103 in sodiumalginate (SA) hydrogel and detected fluorescent proteins secretedinto the liquid medium.
E. coli pTD103 stock was streaked onto LB-Kan agar and incubated at 37 ◦ C 24 −
48 hours as a fresh growth thencultivated into LB-Kan broth and incubated at 37 ◦ C for 4 hours at37 ◦ C, 220 rpm in a benchtop orbital shaker incubator (Grant-bio ™ )prior to a centrifugation at 1 ,
500 rpm for 10 minutes (Sigma 4K15 ™ ,mid bench centrifuge). Cell pellets were harvested, re-suspendedin 2% and 4% SA solution and dropped into 50 mM CaCl solutionto form SA beads. The beads were stored at 4 ◦ C overnight prior tothe setting up of the experiments.On the day of experiment, the beads were washed twice by steriledistilled water then incubated in LB-Kan broth at 37 ◦ C for 72 hourswithout shaking. Supernatants were harvested and centrifuged at20 ,
000 times the gravity force for 20 minutes at 4 ◦ C to remove cellpellets and other residues. This procedure ensures that the detectedmolecular output signal was the one propagated into the media,not inside the cells or any SA residues. The molecular output signalwas detected and recorded by iBright FL1000 system ™ (see Figure6). In this experiment, we utilised SA hydrogel due to its perme-ability of nutrient infusion for cell growth, as well as its usage asa protective layer from environmental hazards [3, 24]. We usedtwo concentrations of SA, 2% and 4%, to observe the cell growthand production of the molecular output signal within the SA beads (a) (b) Figure 6: Transmission of the molecular signal into the liquid medium after 72 hrs incubation. Cells were captured in 4% and 2%SA hydrogel, then incubated with LB-Kan broth for 72 hrs without shaking. The experiment was performed once. The imageswere taken by iBright FL1000 system (Invitrogen). (a) Observation of the spatial distribution of the molecular output signal ina plate. (b) Observation of the molecular output signal production in test tubes, where the engineered bacterial population isplaced at the bottom of the tubes.
Figure 7: Secreted molecular output signals recorded after hours. The cells were encapsulated in agarose hydrogel bub-ble and then incubated with . agarose-Cam for hours.The experiment was performed as triplicates, and the im-ages were taken by iBright FL1000 ™ system (Invitrogen ™ ). and into the liquid medium. As it can be noted from Figure 6a, themolecular output signal propagates with better performance at 4%SA in comparison to the 2% SA. A similar result is shown in Figure6b, where the molecular output signal is shown in the solid and inthe supernatant parts at the bottom and at the top of centrifugetubes, respectively. This suggested that cells secreted fluorescentproteins while being encapsulated within the SA hydrogel beads.This phenomenon was also observed in other studies [15, 29]. Nev-ertheless, we cannot deny a possibility that the cells may haveescaped from the hydrogel beads and produced fluorescent proteinswhile growing in medium.To confirm that E.coli pTD103 can secrete fluorescent proteins[ sfGFP ] into their surrounding environment without escaping, weperformed another experiment with solid medium (i.e., agarosehydrogel). For this experiment, the E.coli pTD103 were cultivated inLB-Kan broth for 3 − . ◦ C, 220 rpm. The cell pellets wereharvested by centrifugation at 1 ,
500 rpm for 5 min (Sigma 4K15 ™ ,mid bench centrifuge), re-suspended then mixed well in warm 1 . ϕ
100 mm petri dishes(Sarstedt ™ ). Once the bacteria-agarose mixture solidified, the petridishes were incubated at 37 ◦ C for 24 hours subsequent steps. Eachbacteria-hydrogel bubble was placed onto a petri dish containing1.5% agarose-Cam and incubated at 37 ◦ C for 24, 48 and 72 hours. At each time point, the fluorescent signals were recorded by iB-right FL1000 system ™ (Invitrogen ™ ). At the 72 hours time point,a triplicate was performed, and the result can be seen in Figure7. The bacteria were entrapped within 1 .
5% agarose gel contain-ing nutrient (LB) as well as antibiotics (Kan) in order to maintaincell growth as well as their ability to produce the desired proteins [ s f GFP ] ). According to our results, after 24 hours incubation, thebacteria grew well and started to produce the molecular outputsignal (data not shown). Once placed into a solid medium contain-ing 1 .
5% agarose-Cam, after 72 hours, a ring of fluorescence wasobserved at the edge of each hydrogel bubble, suggesting the cellscan grow and produce molecular output signal which are diffusedout of their agarose hydrogel bubble (Figure 7). The experimentwas performed with triplicates to ensure its consistency. We elimi-nated the ability for the bacteria to mobilise out of their bubbles byusing Cam, an antibiotic preventing
E. coli pTD103 growth (datanot shown).
The internal machinery of bacteria have been engineered with thepurpose of designing biocompatible technological applications. Inthis paper, we investigated the engineering of a bacterial quorumsensing system to create a positive feedback loop required for thedesign of a bio-nanomachine transmitter. In addition, we proposeto encapsulate the cells in hydrogel to protect them from mixingwith the natural cells in the environment, and this solution canpave the way for intrabody molecular communications systemsusing bacterial signalling in humans and animals.Our numerical analysis shows, considering the scenario inves-tigated, a high throughput with respect to time (reaching morethan 10 − mol/L in 72 hours). It also highlights the saturation ofthe molecular output signal production after 20 hours for most ofthe cases, which can be applied for future biotechnological systemsthat require this stability phase of the bio-nanomachine transmitteroperation. When observing the relationship between the signalpower at the hydrogel edge membrane and the different valuesof molecular diffusion coefficients (i.e., higher or lower viscositymedia), it can be inferred that the hydrogels can facilitate directive propagation, resulting in higher molecular output signal powerthan the conventional free-diffusion propagation.We also provided a proof-of-concept of the bio-nanomachinetransmitter through wet lab experiments, where the bacterial popu-lation is shown to produce higher fluorescence in their close vicinity,and is able to propagate the molecular output signal through thehydrogel medium and into the environment. It is our intention tofurther expand the design introduced here to create an end-to-endmolecular communications systems that can be used for biosensingand biocomputing applications. ACKNOWLEDGMENTS
This publication has emanated from research conducted with thefinancial support of Science Foundation Ireland (SFI) and the Depart-ment of Agriculture, Food and Marine on behalf of the Governmentof Ireland under Grant Number [16/RC/3835] - VistaMilk.
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