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Project description

According to a report from the World Health Organization (WHO), in 2050, each year ten million people will die from pathogenic bacteria that acquired antibiotic resistance if nothing is done.

This is why it is absolutely necessary to find an alternative that can counterbalance this growing antibioresistance. A solution, which is already used in Eastern European countries such as Georgia, is phagotherapy.

This medicine is based on bacteriophages (also nicknamed phages) which are little viruses (from 50 to 200 nm) that can specifically infect a bacterial strain and amplify in it, sometimes leading to the cell death (lytic phages).

This year, the goal of our team is to develop a fully automated system capable of :

• identifying a pathogenic bacteria
• detecting resistance markers
• select the right phages for a possible phagotherapy.

To do this, we will use a DNA probe : a plasmid with a fluorescent gene and a one-stranded fraction which is complementary to the sequence we want to detect. Our automated system will achieve detection from a patient saliva sample. The device will first lyse the bacteria using the phages we are testing and lysis buffer as control. Then, DNA will be extracted using magnetic beads coated with silica. The purified DNA will be digested with the right restriction enzymes to isolate our fraction of interest. The next step will be the hybridization of these fragments with our DNA probes leading to its circularization. Following this, a transformation of the plasmid will be performed into E. Coli competent cells to allow expression of the fluorescence gene and detection.

Our system will be made so the practitioner only has to load samples, run the protocol and come to see the results a few hours later.

For our proof of concept, we will work on two pathogenic bacterial species usually found in nosocomial infection cases due to their ability to develop antibioc resistance : Pseudomonas Aeruginosa and Staphylococcus Aureus. We will make two different probes for each : one for detection of the bacteria, which consists on the recognition of housekeeping genes, and the other for detection of a resistance marker (point mutations on a gene encoding the bacterial wall for example). The two sequences will be detected by fluorescence, using RFP (red fluorescent protein) for housekeeping genes and BFP (blue fluorescent protein) for resistance.

Have a look at our video !

Contact

>igem.grenoble.alpes@gmail.com

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