Team:Grenoble Alpes/Design

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.

WetLab Introduction

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)

Quorum sensing's principle

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.
  

Survival system

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 of BHL.

First, the E7 lysis gene (purple), under the control of the tac promoter, is inserted 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 figure represents the survival system and its functioning according to the presence or absence of BHL.

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 be transcribed 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.

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Therapeutic system

The engineered E. coli will be able to constitutively produce therapeutic molecules, 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).

Figure representing the construction of the therapeutics molecules’ system. The genes of Dispersin B, Alginate lyase, and Pyocin S5 under the control of the J23100 promoter are expressed constitutively.

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 activity which means that it forms pores in its membrane after internalization by an iron transporter specific to P. aeruginosa. The killing domains of Pyocin S5 show homologous domains of the pore-forming colicins Ia and Ib. (A.Parret & R. De Mot 2000)(8)
  
  
• 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. 2008)(9)
  
  
• Alginate lyase is naturally produced by P. aeruginosa which uses it to degrade its 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 disrupt the biofilm and re-expose P. aeruginosa to antibiotics treatments. Furthermore, to avoid any 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.

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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)

The figure represents the delivery system and its functioning. Plsr promoter controls the lysis gene (E7) which is expressed only in the presence of phosphorylated AI-2.

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 be 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 being expressed 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:

• PlsrA promoter : BBa_K649100
• Gene lysis E7 : Bba_K117000 designed by Tokyo_Tech 2011 iGEM team

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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.

DryLab Introduction

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.

Environment module

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.

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Measurement module

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.

Plate Movements

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 requests.

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. In order 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.

References

Team Grenoble_Alpes

Contact

• Igem grenoble
  Place du Commandant Nal
  38700, La Tronche, France

• igem.grenoble.2020@gmail.com

This wiki was completely designed and coded by the team members of the 2020 Grenoble_Alpes' Team, all content on this wiki is available under the Creative Commons Attribution 4.0 license (or any later version).