Cellock Holmes a novel SynBio platform to detect pathogens

Cellock Holmes a novel SynBio platform to detect pathogensAachen 2014Gold MedalBest Supporting Software, OvergradSafety Commendation, OvergradBest Measurement Project

http://2014.igem.org/Team:Aachen/Project1

"What's living on the table in front of you?"Microorganisms

"Whichmicroorganisms are there?

Cellock HolmesCellock Holmes novel biosensor system that detects bacteria on solid surfaces using low cost rapid & portable device WatsOn

This gram-negative prokaryote infects patients with open wounds and burns as well as immunodeficient people.P.aeruginosacells use quorum sensing to communicate with each other by secreting autoinducers into their environment.4Two modulesSensor chips containingCellocks,engineered detective cellsthat fluoresce in the presence of the pathogen, make up the biological part ofCellock HolmesMeasurement deviceWatsOnand the complementarysoftwareMeasurarty- the technical side

Biosensor systems are usually based on a reporter gene under the control of a promoter directly induced by the biomolecule of interest. In the case of our 2D biosensor forPseudomonasaeruginosa, the expression of our fluorescing reporter protein is directly induced by the activity of the bacterium's quorum sensing molecules. However, transcription, translation, folding and post-translational modifications take their time. Since our goal is to detect the pathogen as fast as possible, we aimed to develop a system that provides a fluorescent answer faster than just expressing the fluorescent protein GFP.As an alternative to the traditional approach, weconstitutively express our reporter gene in a quenched form. By fusing GFP to REACh, a mutated variant of EYFP, its original fluorescence is thoroughly suppressed. Thereby, a huge stock of temporary inactivated fluorescence proteins is already available prior to first contact with the pathogen. As a consequence to the uptake of homoserine lactones of P.aeruginosaand binding to the LasI promoter in front of the protease gene, theseautoinducers activate the expression of the TEV protease. This way, our biosensor provides a fast increase in fluorescence intensity by cleavage of GFP-REACh-constructs.WhenP.aeruginosais detected by our cells, the reporter protein has already been expressed and only waits to be activated. The cleavage reaction catalyzed by the TEV protease is a faster process than expression and correct folding of GFP. Therefore, our sensor cells provide an exceptionallyearly response.While a certain concentration of homoserine lactone will produce the same number of gene read-outs and the TEV protease is about the same size as GFP (~27 kDa), one TEV protease can cleave many GFP-REACh constructs per time than fluorescent protein molecules could be expressed and go through fluorescence maturation. Through this additional reaction, we introduce anamplification stepinto our system. Hypothetically, the TEV protease will enable the production ofa much stronger signalin a given time interval.In our project, we reproduced the REACh1 and REACh2 proteins by subjecting an RFC-25 compatible version of the BioBrickE0030(EYFP) to aQuikChange mutation, creating the BioBricksK1319001andK1319002. Subsequently, we fused each REACh protein withGFP (mut3b)which is available as BioBrickE0040. The protein complex was linked via aprotease cleavage site(K1319016). As constitutive promoter we useJ232101. When GFP is connected to either REACh quencher, GFP will absorb light but the energy will be transferred to REACh via FRET and is then emitted in the form of heat; the fluorescence is quenched. Our cells also constitutively express theLasRactivator. Together with the HSL molecules fromP. aeruginosa, LasR binds to theLasIpromoterthat controls the expression of the TEV protease, which we made available asK1319004. When the fusion protein is cleaved by the TEV protease, REACh will be separated from GFP. The latter will then be able to absorb and emit light as usual.

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This flow sheet shows the procedure to sample and detectP.aeruginosa: A sampling chip is briefly put onto the potentially contaminated surface, added onto one of our sensor chips and inserted intoWatsOn.8

Mode of action insideWatsOnChips are incubated at 37C to stimulate cell growth and then illuminated with blue light to excite fluorescence. A picture is taken and analyzed for fluorescence signals using the softwareMeasurarty.9

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