Team:Grenoble Alpes/Proof Of Concept

PyoBuster - Proof of concept

Proof of concept

Our initial objective is to design a genetically modified PyoBusters bacteria able to detect and destroy the Pseudomonas aeruginosa’s biofilm. But also by the design of a testing bench to test our engineered bacteria.

Can we sense the biofilm of Pseudomonas aeruginosa in the infection site ? Can this sensing trigger a specific answer?

FOR SURE!

Our PyoBusters were able to detect BHL, a molecule found in P. aeruginosa’s biofilm infection. We managed to trigger an eGFP expression with synthetic BHL as seen in figure 1.

graphic survival system
Figure 1: eGFP expression according to the BHL concentration and over time. Different concentrations of BHL were performed to evaluate the effect of BHL in E. coli Nissle. Thanks to the BHL, the engineered E. coli should be able to express eGFP. Without BHL, no eGFP expression should be detected.

Thus, thanks to the new part BBa_K3463019, our engineered E. coli Nissle is able to sense low concentration of BHL (1µM) in its environment and express a specific gene under the control of PrhlR. Therefore, we are very confident that the LacI protein could be produced when our engineered E. coli Nissle senses a P. aeruginosa’s biofilm (BBa_K3463031). Associated with the part BBa_K3463028, this LacI production will allow PyoBusters to survive only in the presence of BHL and grow specifically on the biofilm.


Can we use the E. coli own quorum sensing to induce a gene expression?

YES!

We used lsrA promoter to produce mCherry as shown in the figure below. This production depends on the accumulation of AI-2 ( E. coli’s quorum sensing molecule) and therefore on the density of the PyoBusters population. AI-2 is internalized before the stationary phase and this is reflected in a delay in the expression of PlsrA fluorescence compared to the constitutive promoter J23100.

graphic delivery system
Figure 2: mCherry fluorescence expression reported at bacterial optical density over time. To represent the two curves, we used two gradations: the one on the left represents the expression of the lsr promoter and the one on the right represents the J23100 promoter.

We estimate that BBa_K3463025 parts which include the lsrA promoter and the lysis gene e7 we designed will work on the same principle except that lysis will occur instead of fluorescence expression. Indeed the lysis gene e7 has already been tested and approved by other iGEM teams such as NTU-Singapore 2008 iGEM or SZU-China 2019 iGEM team.
We succeeded in producing mCherry thanks to the quorum sensing molecules AI-2 we will as well produce E7 lysis protein according to the PyoBusters population thus permit the delivery on Pseudomonas aeruginosa’s biofilm therapeutic molecules accumulated during the growth.


Have we been able to create a working testing bench?

OF COURSE!

Our incubator should provide a closed and controlled atmosphere as we showed in the results page.

During our experiments, we created disruptions at different moments and the outcome was positive. The prototype proved the performance during at least 12 hours as well as a good ability to re-stabilize after a disturbance such as the opening of the incubator.


Can it reproduce various physiological conditions?

ABSOLUTELY!

The atmosphere is controlled and monitored. It is able to stabilise at any temperature and humidity from the starting parameters to higher values. We focused on recreating the atmosphere's physiological conditions and our environment module perfectly works: in about 20 minutes, it reaches extreme conditions such as 37°C (310°K) and 95% of relative humidity. This atmosphere is stable and homogeneous inside the incubator. We also showed that the atmosphere inside a covered plate was also reaching the desired temperature and the desired humidity in about 30 minutes.

graphic shows the temperature and humidity evolution
Figure 3: results of the environment module on a 30 minutes experiment

Figure 3 is associated with the previous example of reaching 37°C and 100% of relative humidity. It shows the data of the 6 sensors that measures the atmosphere inside our box. Sensor number 5 is inserted inside a covered plate to understand how temperature evolves inside. This graph shows that whether the plate is covered or not, our environment module provides the desired temperature within 30 minutes and humidity reaches 95%. Atmosphere is stable and we let it run for 12 hours and it stayed at the desired temperature and humidity.


How does the testing bench see the evolution of biological samples?

Through a fluorescence detection module described in Hardware, we can monitor 2 different fluorescence in order to follow both biofilms and E. coli populations in a 96 well plate. In the following figure 4, you can see the pictures of samples containing E. coli strained with eGFP.

fluorescence well 1 fluorescence well 2 fluorescence well 3 fluorescence well 4
Figure 4: pictures of wells taken by the picamera

The values obtained after calculation are the average intensities of the pixels of interest corresponding to the fluorescence emitted by the sample. You can see in figure 5 the images converted in binary images, and with these images we are able to recover the pixels of interest. The values we obtained allow us to quantify the fluorescence emitted and we compared these results with the industrial plate reader FLUOstar Optima from BMG LABTECH in order to validate the fluorescence module.

binary fluorescence well 1 binary fluorescence well 2 binary fluorescence well 3 binary fluorescence well 4
Figure 5: Binary images obtained from the previous 4 pictures.

How was plate agitation implemented?

We recreated in 2D the movement hardware of 3D printers. It means that the plate is able to be anywhere in a square of 15cm*15cm by steps of 0,2 mm which is quite precise. The speed of rpm is linked to our motor specifications and we were able to recreate an agitation up to 120rpm associated with a diameter of the agitation of 4cm. This video explains everything that we implemented for plate movements.


Do we have a friendly human-machine interface?

WITHOUT ANY DOUBT!

We created our interface that is both natural and convenient to use. You can choose all the parameters you need for a cell growth experiment and the products you insert inside the selected wells. The interface keeps in memory every experiment done previously and you can select them to pre fill the same conditions.

Another strong point is the possibility to create pauses on the experiment, where you are able to change the settings or add new products inside the plate.

We made a video tutorial of our interface that explains how to use it.


How does the communication inside our hardware system work?

THANKS TO the HermAs protocol!

Our self made incubator is composed of 3 hardware modules that are controlled by our software: atmosphere control, plate movements and fluorescence detection. It is composed of 4 small computers, one for each module and the last one is the masterchief.

One major side of our software system is the communication between all the devices. HermAs protocol is a communication adaptable to any hardware system and allows a modular hardware system. Hence, each module has its own hardware and software system which means they work independently.

This video shows how the HermAs protocol works.