Latest revision as of 09:25, 12 September 2018

The goal of our detector is to reveal the presence of Pseudomonas aeruginosa in a sample thanks to the specific lysis by a bacteriophage. The design of this detector has been inspired by Cork Ireland 2015 team : they created different based on the perfect complementarity of the double strand DNA.
Thus, we - Grenoble team 2018 - decided to create a similar detector for Pseudomonas aeruginosa pathogen.

1. Bioinformatics Part

1.a. Choice of the target

The detection of bacterial lysis will be done by detecting a gene fragment. Indeed, the gene fragment is intracellular so when detected it implies that lysis occurred.
With this in mind, some reading was done, first, on how to characterize the lysis of Pseudomonas aeruginosa by bacteriophages. At first, we decided to work on virals factors genes. However most of it are find in another strain than Pseudomonas aeruginosa.
Housekeeping genes are genes that are required for the maintenance of basic cellular function. They are normally expressed in all bacterias 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. ProC is an housekeeping gene found in all Pseudomonas aeruginosa strains and not linked in pathogenic behavior of Pseudomonas aeruginosa. Indeed, in 2003, Hakan Savli et al. [1] 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 could conclude that this pair could be used as internal controls in relative comparison studies of resistance genes in P. aeruginosa.
Once the gene is selected, fraction of it were inserted in NebCutter, to get its natural restriction sites. So that we could select several fragments nucleotide (less than 100bp) cut in blunt ends by restriction enzymes.
These fragments are entered in the NCBI database for sequence alignment. By trying several fragments we chose the one that were the most specific to PAO1. Finally, the target was found in ProC gene (822bp) from PAO1 strain (GenBank : AAG03782.1) located in PA0393 locus. The target is located between nucleotide 766 and nucleotide 802.

1.b. Design of the probe

The probe aims to receive the target, by binding by perfect complementarity. It is made by :
→ Two restrictions enzymes producing cohesives and, SphI and NgoMIV, which goal is to remove the little sequence in between on the bottom strand and thus create a perfect complementarity with the target. But at the end we trust that using PCR linearization could reduce the background of uncut plasmid.
→ Twi nicking enzymes, Nt.BspQ1 and Nb.Bsm1, enzymes that cut one strand of the double DNA strand. Thereby, the top strand is removed, allowing the binding of the target.

1.c. Choice of the plasmid backbone

We chose a plasmid from the iGEM 2018 DNA distribution kit, following different conditions to screen. We wanted the plasmid to :
→ have a red fluorescent reporter: RFP or mCherry
→ high level copy
→ properly sequenced
→ have no restriction sites that were used for the probe
→ not be arabinose inducible (the first tests we did were not conclusive) so preference for IPTG induction

1.d. New part : BBa_K2629000

Then, the part we designed is made with two parts : the detector, i.e. the probe and the plasmid backbone, carrying a reporter.
To sum up, once the probe is activated/digested, a “window” is opened: a short part of the detector is single strand, allowing the ligation of the target by complementarity and then the recircularization of the plasmid, which can be transformed.

2. Practical Part

How to insert the probe into the plasmid psB1C3-BBa_J04450 ?

References

[1] Expression stability of six hausekeeping genes : A proposal for resistance gene quantification studies of Pseudomoas aeruginosa by real-time quantitative RT-PCR, Savli H, Journal of Medical Microbiology 52(pt : 5) : 403-8, June 2003