Wet Lab Design
PyoBusters is a genetically engineered E. coli strain, fulfilling a therapeutic action against P. aeruginosa and its biofilm. Our PyoBusters system works thanks to a plasmid comprising 4 different sets of genes, as well as a gene inserted in the genome.
Those sets of genes can be separated into 3 systems, operating in parallel with different objectives.
- Survival system: Specifically target the P. aeruginosa’s biofilm to grow on its surface.
- Therapeutic system: Produce intracellular therapeutic molecules to destroy P. aeruginosa and its biofilm.
- Delivery system: Work as a delivery system allowing the in-situ release of therapeutic molecules.
The core of PyoBusters is based on quorum sensing (QS). The latter is an aptitude used by most bacteria living in a community to regulate many genes. When bacteria growth reaches a density threshold, bacteria can communicate together to regulate the expression of specific genes in their genome. Thereby, thanks to the QS, bacteria can modulate their behavior according to their environmental density. (S. Mukherjee & B. L. Bassler 2019)(1)
PyoBusters uses two different QS molecules:
- The first one is the own quorum-sensing of our engineered E. coli, namely Autoinducer 2
(AI-2) which would be rerouted to serve our purpose (J. Li et al. 2007)(2). These
molecules will be used for the delivery system and will regulate the growth of
PyoBusters on the biofilm.
- The second one is BHL (C4-HSL). These quorum-sensing molecules are specific to
P. aeruginosa and detectable in many cases of biofilm infections (M. Hentzer et al.
2003)(3). Thus, BHL will be used to allow PyoBusters to sense the P. aeruginosa’s
biofilm in vivo. However, as it is not a molecule naturally used by E. coli, we have to
insert all the required DNA systems into our engineered bacteria.
This part of the PyoBusters system focuses on the BHL sensing and its importance for both specificity and safety of our innovative treatment. Indeed, the presence of this small diffusible molecule allows PyoBusters to detect the P. aeruginosa’s biofilm and therefore induces its survival (M. Hentzer et al. 2003)(3). However, without BHL in its environment, PyoBusters will not be able to grow and die. Thus, this cascade ensures that the PyoBusters’ growth will only be possible on the biofilm of P. aeruginosa, without colonising the entire environment.
To activate our cascade system, we use a specific interaction between the BHL and the
transcriptional activator RhlR (N. Chowdhury et al. 2018)(4). Through specific interactions
with BHL, RhlR can change its conformation and become effective to bind a specific promoter
sequence (Prhlr). Thus, all genes downstream of Prhlr can be expressed
only in the presence
First, the E7 lysis gene (purple), under the control of the tac promoter, is
instead of the lacI gene into the E. coli genome to obtain an E.
coli DH5 ΔLacI Ptac-E7.
Then, this engineered strain is transformed with a plasmid that contains two DNA sequences:
J23100-RhlR (red) and PrhlR-LacI (yellow).
The parts used to engineer this strain are the following:
- Constitutive promoter (red): BBa_K3463008
- PrhlR promoter (yellow): BBa_K3463031
- Ptac promoter (purple): BBa_K3463028
(A) Without BHL in the medium, the E7 lysis protein is constitutively produced by the
bacteria because no repression by the LacI protein occurs. Thus, the lysis of the bacterial
membrane will be triggered and PyoBusters will die.
(B) When the quantity of BHL is high enough in the environment of PyoBusters, these small
diffusible molecules can diffuse through the bacterial membrane to interact with the RhlR
protein, already constitutively produced in the cytoplasm by the J23100 promoter. This
interaction leads to the formation of an active transcriptional complex that is able to bind
the rhlR promoter. Thanks to the activation of PrhlR, the LacI protein can
and produced to inhibit the lysis protein expression in the genome via the repression of the
tac promoter. PyoBusters can then grow on the biofilm.
The engineered E. coli will be able to constitutively produce therapeutic
namely Dispersin B (DspB), Alginate Lyase (AlgL), and Pyocin S5 (PyoS5). They are
expressed through a constitutive promoter and will accumulate in the cytoplasm during
bacterial survival. Two of these proteins, namely Dispersin B (I. Y. Hwang et al. 2017)(5)
and Alginate lyase, are capable of degrading the biofilm (B. Zhu & H. Yin 2015)(6) and the
Pyocin S5 is aimed to kill P. aeruginosa thanks to a bactericidal effect (Y.
Michel-Briand & C. Baysse 2002)(7).
To realize the therapeutic system we used the following parts:
- Dispersin B: BBa_K1659200, designed by Wei Chung Kong iGEM15_Oxford15 (2015-08-28)
- Alginate lyase: BBa_K3463002
- Pyocin S5: BBa_K3463001
Pyocin S5 is produced by P. aeruginosa. It presents channel-forming
means that it forms pores in its membrane after internalization by an iron
transporter specific to P. aeruginosa. The killing domains of Pyocin S5
homologous domains of the pore-forming colicins Ia and Ib. (A.Parret & R. De Mot
Dispersin B is an exo-acting glycosyl hydrolase that catalyzes the hydrolysis of
polymers constituting the biofilm especially on the β(1,6) linkages of
N-acetylglucosamine (GlcNAc) present in P. aeruginosa’s biofilm. (J.
Kerrigan et al.
Alginate lyase is naturally produced by P. aeruginosa which uses it to
biofilm, mainly constituted of alginate. Therefore, its utilization as a biofilm
reducer is more than relevant. (H.Tavafi et al. 2018)(10)
These molecules have been chosen to have a considerable impact on the biofilm and the
P. aeruginosa population. Indeed, the Dispersin B and the Alginate lyase will
biofilm and re-expose P. aeruginosa to antibiotics treatments. Furthermore, to
dispersion of the biofilm that could lead to a new infection, the Pyocin S5 is used to
lyse P. aeruginosa once they are vulnerable.
Thus, this therapeutic cocktail would allow the PyoBusters system to fight efficiently
against the infection by releasing these molecules on the biofilm during the activation
of its delivery system.
The delivery system is an essential part of PyoBusters which allows therapeutic molecules to be released on the targeted biofilm. This system is based on E. coli’s QS and more specifically Autoinducer 2 (Ai-2). Indeed, Ai-2 is constitutively produced and secreted by E. coli and interacts with endogenous proteins like LsrR. (J. Li et al. 2007)(2)
LsrR is a protein containing a binding site that possesses a high affinity for lsr promotor and ordinarily inhibits the transcription of the operon. When the E. coli growth reaches a stationary phase, a massive internalization of Ai-2 occurs. Once in the cell, Ai-2 is phosphorylated and blocks the binding of LsrR on the lsr promoter. Thus, the repression by LsrR is inhibited and the transcription of Plsr downstream genes occurs. (M. O. Din et al. 2016)(11)
In our system (green), we fused the lysis gene E7 downstream the
lsrA promoter. Thus
the expression of lysis protein (colicin E7) under the control of Plsr will
triggered when the density of PyoBusters will be high enough on the biofilm leading
to E. coli lysis. In addition, lsrA promoter has the advantage of
only at the beginning of the stationary phase which allows a large number of
therapeutic molecules accumulated in the cytoplasm during the growth to be released.
(C. E. Torres-Cerna et al. 2019)(12)
This system not only permits the delivery of therapeutic molecules but also enables the growth regulation of E. coli’s populations.
To realize the delivery system we used the following parts:
Dry Lab Design
PyoBusters’ goal is to destroy the biofilm of P. aeruginosa which acts in different environments. As engineers we wanted to provide the experimenters a way to grow their biofilms and insert the modified E. coli in an automated experience. Hence we built an incubator called the Automated Measurement Incubator (AMI) which is built and implemented in 4 parts:
- The environment module that controls and monitor the atmosphere inside the box.
- The agitation module that operates the plate movements.
- The fluorescent module that makes the analysis during the whole experiment.
- The software that controls the 3 previous modules and allows a Human Computer Interface.
When you do cell growth experiments, you need 2 devices: one incubator and one fluorescence microscope. AMI is specifically done for cell growth experiments where you should be at this precise temperature and humidity with fluorescent analysis. Our incubator recreates different atmospheres by adapting temperature and humidity, agitating the plate at a certain rpm and analysing each desired well with the detection module. AMI Is so a complex and complete tool and its last strong point is its price because it costs less than 1000 euros.
One of the first requirements was to control two environmental parameters inside a
closed box: the temperature and the humidity.
Concerning the temperature, we choose to use heating resistors. It allows us to reach
any desired temperature that is above the surrounding temperature. We concentrated on
using only heating resistors to simplify the first build-up and if it was working by
heating the atmosphere, it would also be working by cooling the atmosphere thanks to the
Peltier modules. We didn’t have the time to add the Peltier modules so we won’t talk of
this unless it is in the enhancement part.
Concerning the humidity, the idea of heating a water tank would complexify our system
because water may be hotter than the desired temperature, and it would be difficult to
implement the impact of the water tank on the atmosphere. To create high evaporation,
maybe we would need a big water tank that would create high heat exchanges with the
atmosphere. Then, we came up with the idea of an ultrasonic atomizer which was simpler:
you atomize water into microdrops independently from its temperature. We calculated that
we would need a few grams of water inside the room to reach 100% relative humidity,
hence the water tank of the water atomizer doesn’t need to be that big.
Concerning sensors, our choice went on the DHT 22 because of their good accuracy,
stability over time and ease of communication with Arduino.
To follow the evolution of the biological system into the multiwell plate, we had to
adapt our measurement system to the different biological constraints: the space, the
microvolumes to observe, the monitoring of both biofilms and therapeutic molecules in
the same environment.
Firstly, we chose to monitor the evolution of the biological system using fluorescence.
This technique requires high precision especially when we have to deal with low volumes.
The choice of the emission and absorption filters specific to our two fluorescence
spectra (eGFP and mCherry) is crucial because the intensity of the fluorescent signal
coming from the plate is weak. Moreover, to be able to focus the excitation light very
close to the well to be observed and on the other side to collect as much emission light
as possible, we designed a 2D system capable of moving the 96 well plate. With this
system, we can focus on a single well to be as close as possible from the measures
obtained using a regular fluorometer. The device which collects the light is a camera
connected to the Raspberry and after acquiring a picture of the sample, an image
processing program deals with the picture to extract the information of interest and
delete the small artefacts.
As explained in the previous part, to deal with the low volumes of culture in 96 well
plates, we built a system able to move this plate in a 2D horizontal plane taking
inspiration from the 3D printer we used.
Furthermore, another component of the culture growth is the agitation needed to improve
the growth. With the 2D moving plate system, we can also agitate in a proper way the
solutions in the 96 well plate by modifying the program which controls the motors.
Devices communication and interface
All our system is controlled by four programmable microcontrollers (3 Arduino, 1
Raspberry). Each one has a specific action in our system: the first Arduino controls the
temperature and the humidity, the second one manages the plate movements, then the last
Arduino deals with the fluorescence system and finally the Raspberry controls the
camera. Microcontrollers communicate with each other following a certain hierarchy. The
Raspberry has the master role and sends the orders and the requests to the different
Arduino microcontrollers that execute the commands, and reply to the Raspberry’s
Concretely the data between the master and slaves transit through a USB connection
thanks to our own communication protocol: HermAs. With this protocol, the Raspberry
controls perfectly all the data needed for the good efficiency of the microcontrollers.
for users to control the system we implemented a web server which is run by the
Raspberry. By logging on the interface, they can start and stop an experiment and follow
it easily and quickly throughout all the process.
- Mukherjee S, Bassler BL. Bacterial quorum sensing in complex and dynamically changing environments. Nat Rev Microbiol. 2019 Jun;17(6):371-382. doi: 10.1038/s41579-019-0186-5. PMID: 30944413; PMCID: PMC6615036.
- Li J, Attila C, Wang L, Wood TK, Valdes JJ, Bentley WE. Quorum sensing in Escherichia coli is signaled by AI-2/LsrR: effects on small RNA and biofilm architecture. J Bacteriol. 2007 Aug;189(16):6011-20. doi: 10.1128/JB.00014-07. Epub 2007 Jun 8. PMID: 17557827; PMCID: PMC1952038.
- Hentzer M, Wu H, Andersen JB, Riedel K, Rasmussen TB, Bagge N, Kumar N, Schembri MA, Song Z, Kristoffersen P, Manefield M, Costerton JW, Molin S, Eberl L, Steinberg P, Kjelleberg S, Høiby N, Givskov M. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors. EMBO J. 2003 Aug 1;22(15):3803-15. doi: 10.1093/emboj/cdg366. PMID: 12881415; PMCID: PMC169039.
- Hwang, I., Koh, E., Wong, A. et al. Engineered probiotic Escherichia coli can eliminate and prevent Pseudomonas aeruginosa gut infection in animal models. Nat Commun 8, 15028 (2017). https://doi.org/10.1038/ncomms15028
- Zhu B, Yin H. Alginate lyase: Review of major sources and classification, properties, structure-function analysis and applications. Bioengineered. 2015;6(3):125-31. doi: 10.1080/21655979.2015.1030543. Epub 2015 Apr 1. PMID: 25831216; PMCID: PMC4601208.
- Michel-Briand Y, Baysse C. The pyocins of Pseudomonas aeruginosa. Biochimie. 2002 May-Jun;84(5-6):499-510. doi: 10.1016/s0300-9084(02)01422-0. PMID: 12423794.
- Parret A, De Mot R. Novel bacteriocins with predicted tRNase and pore-forming activities in Pseudomonas aeruginosa PAO1. Mol Microbiol. 2000 Jan;35(2):472-3. doi: 10.1046/j.1365-2958.2000.01716.x. PMID: 10652108.
- Kerrigan, J., Ragunath, C., Kandra, L., Gyémánt, Gy., Lipták, A., Jánossy, L., … Ramasubbu, N. (2008). Modeling and biochemical analysis of the activity of antibiofilm agent Dispersin B. Acta Biologica Hungarica, 59(4), 439–451. doi:10.1556/abiol.59.2008.4.5
- Tavafi, H., Ali, A. A., Ghadam, P., & Gharavi, S. (2018). Screening, cloning and expression of a novel alginate lyase gene from P. aeruginosa TAG 48 and its antibiofilm effects on P. aeruginosa biofilm. Microbial Pathogenesis. doi:10.1016/j.micpath.2018.08.018
- Din MO, Danino T, Prindle A, Skalak M, Selimkhanov J, Allen K, Julio E, Atolia E, Tsimring LS, Bhatia SN, Hasty J. Synchronized cycles of bacterial lysis for in vivo delivery. Nature. 2016 Aug 4;536(7614):81-85. doi: 10.1038/nature18930. Epub 2016 Jul 20. PMID: 27437587; PMCID: PMC5048415.
- Torres-Cerna CE, Morales JA, Hernandez-Vargas EA. Modeling Quorum Sensing Dynamics and Interference on Escherichia coli. Front Microbiol. 2019;10:1835. Published 2019 Aug 20. doi:10.3389/fmicb.2019.01835