Team:Grenoble-Alpes/Demonstrate
Our results
The Aiguilles d’Arves are not only the apex the Maurienne valley, they are also its emblem. Credits: Estelle Vincent
Main results - The proof of concept - Improvements
Main results
SnapLab project was divided into five main missions : establishing an extraction process to get the target, constructing the DNA detector, finding solutions for the conservation of the biological content within the kit, creating a detection system using a smartphone to give a diagnosis only with the portable kit and adapting the kit to biological constraints in parallel of its modelisation.
About the target preparation
About the extraction, the problematic was the following: how to extract, from a stool sample, the small DNA target sequence ? Investigations were done about Vibrio cholerae’s functioning. How does it grow in a laboratory ? What are the tanks, how does contamination happen ? What is its physiopathology ? In parallel, current techniques for DNA extraction were reviewed. Finally, a few protocols for Vibrio cholerae (and especially CTX genome) extractions were established thanks to these data. For safety reasons, practical tests were done on E. coli bacteria (strain: DH5α). Also, some parts of the protocol were done in kit-like conditions. For example, steps for which liquids had to go through a silica membrane were done thanks to a piston, to avoid centrifugation, since it would not be possible within the kit. Results were very promising and the main conclusions are : (i) extracting genomic DNA without centrifugation is possible and (ii) elution with TE buffer gives better concentrations than with water.
At the end of the extraction process, Vibrio cholerae DNA is theoretically digested with AluI enzyme, then the targeted sequence is isolated and denatured. At this stage, the target is ready to be detected!
About the construction of the plasmid detector
The second biological mission was the construction of the plasmid detector. Inspired by the genius of team, who designed an amazing DNA detector for Human Papillomavirus, Grenoble Alpes 2017 team created a similar biological system to detect the specific target sequence of Vibrio cholerae (more precisely: 39bp found in RstA gene of bacteriophage CTX). A probe was designed according to the target sequence, and inserted into an appropriate plasmid backbone carrying a fluorescent reporter gene. This insertion was more complicated than expected. The main conclusions for this mission are :
- (i) an insertion of sequence inside a plasmid vector through a unique restriction site requires at least three dephosphorylation steps to avoid self-ligation
- (ii) the best ratio insert:vector in that case appeared to be a molar ratio 1:1
- (iii) an insertion between two different restriction sites is much easier than one in a unique site
The constructed detector was activated and then tested with the target, with successful results. Few false positives were observed but were suspected to be due to a bad digestion of the detector probe prior to the contact with the target. Still, there is a notable difference of colonies’ number when the target is present or not. Evaluation of sensitivity and specificity was also attempted. Sensitivity, which is the limit of detection of the test, was tested through the counting of transformed bacteria. An important difference between absence or presence of the target was observed as soon as 6ng of target was in contact with 100ng of detector. However, a sequencing of all colonies would have been more accurate, to ensure that the counting was not taking false positives into account. Specificity, which is the proportion of positives identified as such, brought more issues. The tests consisted in the evaluation of the detector’s ability to recognize only the original target and not the false sequences more or less homologous. Unfortunately, results were too heterogeneous to draw any conclusions.
As a conclusion, a new biobrick was built for which the proof of concept for its ability to detect cholera’s pathogen DNA was successfully achieved. However, its characterization should be completed by statistical analyses.
About the detector and bacteria’s conservation
About the detection
About the kit itself
The proof of concept
Improvements
About the target preparation / About the construction of the plasmid detector / About the detector and bacteria’s conservation / About the detection / About the kit itself
Cyprum itidem insulam procul a continenti discretam et portuosam inter municipia crebra urbes duae faciunt claram Salamis et Paphus, altera Iovis delubris altera Veneris templo insignis. tanta autem tamque multiplici fertilitate abundat rerum omnium eadem Cyprus ut nullius externi indigens adminiculi indigenis viribus a fundamento ipso carinae ad supremos usque carbasos aedificet onerariam navem omnibusque armamentis instructam mari committat.
Thalassius vero ea tempestate praefectus praetorio praesens ipse quoque adrogantis ingenii, considerans incitationem eius ad multorum augeri discrimina, non maturitate vel consiliis mitigabat, ut aliquotiens celsae potestates iras principum molliverunt, sed adversando iurgandoque cum parum congrueret, eum ad rabiem potius evibrabat, Augustum actus eius exaggerando creberrime docens, idque, incertum qua mente, ne lateret adfectans. quibus mox Caesar acrius efferatus, velut contumaciae quoddam vexillum altius erigens, sine respectu salutis alienae vel suae ad vertenda opposita instar rapidi fluminis irrevocabili impetu ferebatur.
Et eodem impetu Domitianum praecipitem per scalas itidem funibus constrinxerunt, eosque coniunctos per ampla spatia civitatis acri raptavere discursu. iamque artuum et membrorum divulsa conpage superscandentes corpora mortuorum ad ultimam truncata deformitatem velut exsaturati mox abiecerunt in flumen.
Improvements
Fieri, inquam, Triari, nullo pacto potest, ut non dicas, quid non probes eius, a quo dissentias. quid enim me prohiberet Epicureum esse, si probarem, quae ille diceret? cum praesertim illa perdiscere ludus esset. Quam ob rem dissentientium inter se reprehensiones non sunt vituperandae, maledicta, contumeliae, tum iracundiae, contentiones concertationesque in disputando pertinaces indignae philosophia mihi videri solent.
Superatis Tauri montis verticibus qui ad solis ortum sublimius attolluntur, Cilicia spatiis porrigitur late distentis dives bonis omnibus terra, eiusque lateri dextro adnexa Isauria, pari sorte uberi palmite viget et frugibus minutis, quam mediam navigabile flumen Calycadnus interscindit.
Nisi mihi Phaedrum, inquam, tu mentitum aut Zenonem putas, quorum utrumque audivi, cum mihi nihil sane praeter sedulitatem probarent, omnes mihi Epicuri sententiae satis notae sunt. atque eos, quos nominavi, cum Attico nostro frequenter audivi, cum miraretur ille quidem utrumque, Phaedrum autem etiam amaret, cotidieque inter nos ea, quae audiebamus, conferebamus, neque erat umquam controversia, quid ego intellegerem, sed quid probarem.
Nisi mihi Phaedrum, inquam, tu mentitum aut Zenonem putas, quorum utrumque audivi, cum mihi nihil sane praeter sedulitatem probarent, omnes mihi Epicuri sententiae satis notae sunt. atque eos, quos nominavi, cum Attico nostro frequenter audivi, cum miraretur ille quidem utrumque, Phaedrum autem etiam amaret, cotidieque inter nos ea, quae audiebamus, conferebamus, neque erat umquam controversia, quid ego intellegerem, sed quid probarem.
Adolescebat autem obstinatum propositum erga haec et similia multa scrutanda, stimulos admovente regina, quae abrupte mariti fortunas trudebat in exitium praeceps, cum eum potius lenitate feminea ad veritatis humanitatisque viam reducere utilia suadendo deberet, ut in Gordianorum actibus factitasse Maximini truculenti illius imperatoris rettulimus coniugem.
