Team:Grenoble-Alpes/selection
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TARGET SELECTION
How do we do this?
If the DNA is detected, it means that the plasmid re-circularized itself and is ready to be transformed into E.coli in order to produce fluorescence. Finally, if the system detects fluorescence it means that the lysis has been done.
Thus, the first step is to select the target that characterizes the lysis and resistance markers of P. aeruginosa. We choose to take a fragment of DNA to characterize the lysis, since it will be detectable only if the DNA is released. Regarding resistance markers, we focused on genes that are often the cause of it.
These fractions will be the targets of our detection probes.
PSEUDOMONAS AERUGINOSA DETECTION
In Pyo, the first studies were possible thanks to the sequencing of the complete genome of the pathogenic strain PAO1 [1], this strain is considered to be the reference strain. This is why in our project, we chose to use this bacterial strain in order to prove the concept of our system.
Target that characterizes the lysis of Pyo
The detection of a bacterial lysis will be done by recognizing a gene fragment. Indeed, the gene fragment is intracellular, so when it is detected, it implies that the lysis occurred.
With this in mind, some reading was done on how to characterize the lysis of Pseudomonas aeruginosa by bacteriophages. At first, we decided to work on viral factors genes. However, most of them are also found in other strains than Pseudomonas aeruginosa. We were working on PAO1 strain.
Housekeeping genes are genes that are required for the maintenance of basic cellular functions. They are normally expressed in all bacteria from one strain. We were looking for genes that would be present in most, perhaps even all Pseudomonas aeruginosa. So we choose to focus on this type of genes.
The housekeeping gene used is ProC. Indeed, in 2003, Hakan Savli et al. [3] study “showed that proC and rpoD form the most stable pair in a set of clonally unrelated P. aeruginosa strains with diverse resistance phenotypes.” Moreover, they concluded that this pair could be used as internal controls in relative comparison studies of resistance genes in P. aeruginosa.
Once the gene found, it was inserted into NebCutter to look at the naturally occurring restriction sites. The goal was to find a sequence of about 20-100 nucleotides. This sequence would become the target that we want to detect.
The second objective was to find a specific fragment of Pseudomonas aeruginosa. For this, these fragments are entered in the NCBI database for sequence alignment to judge their relevance. The parameters of the BLAST are : Excluded “Pseudomonas aeruginosa group” and “Highly similar sequences, megablast”.
By trying several fragments we chose the one that were the most specific to PAO1.
The result (Fig. 1) shows a 100% homology with Pseudomonas sp. AK6U. The AK6U strain of P.A produces rhamnolipid biosurfactants in varying degrees when grown on MgSO 4 [3]. This strain is found in soils contaminated with used lubricating oil, benzene and diesel and not in clinical cases.
This result isn’t problematic as the specificity will be provided by the phage. Indeed, a phage is specific of a bacterial strain, so a screening of phages will be done before their use in the device. Finally, the target found in ProC gene (822bp) from PAO1 strain (GenBank : AAG03782.1) is located in PA0393 locus. The target is located between nucleotide 766 and nucleotide 802.
5’- CCCTGAACGCCGCCAGCCAGCGCTCCGCCGAGCTGG-3’
As for the enzyme surrounding the sequence on both sides it is HaeIII.
All this work has been carried out in order to select the target after extracting Pyo's DNA. Indeed, after the extraction, HaeIII digestion during 15 minutes at 37 ° C in CutSmart Buffer will be performed. The target is now ready to be detected and the next step, the detector activation, can occur.