The main goal of our project is to destroy the biofilm of P. aeruginosa. The wet lab that creates a modified E. coli which should destroy the biofilm. The dry lab that creates a benchmark for testing our modified E. coli. On this page, we will present one important step: first the detection of the biofilm by E. coli, and second the environment module of the benchmark.
How to detect P. aeruginosa’s biofilm in a specific manner?
During the development of our project we stumbled on a problem: how to detect P. aeruginosa’s biofilm in a specific manner? Indeed, biofilms of different bacteria have a lot of similarities and therefore it is difficult to create a specific therapeutic device for P. aeruginosa. We needed to think of other molecules or physical parameters that could indicate the presence of the pathogen at the biofilm life stage that wasn’t the biofilm itself. This is how we got to the quorum sensing of the bacteria. Since some molecules of Pseudomonas quorum sensing are specific to the pathogen and are secreted in particular environmental conditions, it was the perfect solution to not only specifically target P. aeruginosa but also target a specific stage of the biofilm formation and avoid spreading elsewhere. As the quorum sensing molecule controls the expression of P. aeruginosa genes, we recovered the quorum sensing gene regulation mechanisms of the pathogen and put our construction under their control.
As our system involved multiple cascades that aim to activate the expression of a lysis gene, it was important that each actor of the cascades is expressed in a sufficient amount. We needed a system to modulate the expression of every gene involved in the lysis of our bacteria, in a way that it would be activated at a certain threshold. One solution could have been to design promoters with different expression levels, but as we had inducible promoters it wasn't ideal. This is why we thought of designing RBSs with different 5' UTRs for all the actors. It allowed us to use our inducible promoters and control the expression through translations and not through transcription. Using these RBSs, we found a combination that triggered the lysis of our bacteria at the desired threshold. As a consequence, if our device is used in different conditions with different sensing conditions, we wouldn't have to redesign our constructions, we would just change the RBSs.
When the constructions were determined and our bacteria was ready to be tested on the biofilm of P. aeruginosa, we stumbled upon the problem of following our bacterial population through the lysis cycles and quantifying the biofilm destruction. This is why we added a constitutively expressed fluorescence gene in our PyoBusters and a camera as well as appropriate fluorescence filters into our testing bench. The biofilm would be coloured with crystal violet and our PyoBusters would express GFP, so we could visually follow their real time evolution in the testing cell without having to remove the cultures from the infection conditions for measures.
How could we create a stable atmosphere inside an incubator?
In our project we created a benchmark for bacteria growth experiments and it also illustrates the different steps of the cycle “Research - Imagine - Build - Test - Learn - Improve - Research”. The dry lab project is made of 4 parts: the environment module, the agitation module, the fluorescent module and the software that operates the 3 modules. The environment module perfectly illustrates the cycle.
We made research to understand what had been done before us and we created the very first outlines of our box. To create a stable atmosphere with the desired temperature and humidity, we thought about building a hardware with a primary and a secondary room. Temperature and humidity would be created in the secondary room and thanks to fans it would be sent to the primary room where the experiment would take place. Though we never build this idea because of the discovery of a new device: the ultrasonic water atomizer. It simplified a lot the creation of the humidity parameter that we built everything in one box. You can see on the figure our first prototype which is only the environment module.
As a first prototype, we chose this box because it was easier than building our own box with its impermeable cover (not on the photo). This prototype is made of sensors (numbered in white from 0 to 5, number 5 is under the plate), heating resistors (the 6 orange parallelepipeds), fans (2 faces one each other), the 96 wells plate for cell cultures and hidden under the right fan there is the humidity module. Outside the box, you can see the microcontroller (one arduino) and the white plate is for electronic wire connections.
To put it simply: an external 20V electronic supply is powering the 6 heaters. Thanks to an electric relay, the microcontroller can manage the heater supply. The 6 sensors allow the microcontroller to monitor what is happening inside the box. Thanks to this prototype, we were allowed to nicely monitor and control our box. The usb connection is for the pc connection. Our first box goes with its own software implemented on the Arduino. Based on a PID corrector, this code should ensure a smooth monitoring and controlling of our box.
Now, the “test step” can begin and we will discover if we can really control the atmosphere inside our box and how it evolves. You can see the results on the next graphes. The experience was to reach 37°C (310°K) and 100% of relative humidity.
This figure tells us everything that we need to understand our prototype. The different graphs show humidity and temperature datas for the 6 sensors, the duty cycle chosen by the Arduino code and the order of the brumisateur. All abscisses are the time in seconds.
Sensors are placed at different locations inside the box and you can see that all sensors show the same result: around 20 minutes, the box has a stable atmosphere at the desired temperature. Humidity is still in process but it is around 96% of relative humidity. The duty cycle of our code is also stable, it means that the permanent system is reached: it is heated enough to compensate for the loss. The brumisateur is a boolean value, it is either 1 for on and 0 for off. It says that it hasn’t reached the desired humidity value but we are higher than 95% which is really nice.
The “test step” is going on with many other experiments to understand how our system reacts for longer duration, when there are disruptions such as opening the cover, or trying other PID corrector values.
The “improve step” is our actual hardware: the Automated Measurement Incubator (AMI). Too see the hardware page click here.