Team:INSA-UPS France/Results

Croc'n Cholera
iGEM UPS-INSA Toulouse 2017

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Results

Texte d'intro

1. Mimicking Vibrio sp. presence with an engineered E. coli

Background

For safety reasons, we cannot manipulate V. cholerae. Therefore to mimic its presence, we engineered E. coli to produce a quorum sensing molecule from a non-pathogenic Vibrio species i.e. C8-CAI-1 from V. harveyi ( cloning ). C8-CAI-1 is an analogue of V. cholera CAI-1 quorum sensing molecule and is produced by the enzyme CqsA. Besides, it allows to investigate our capacity to create synthetic communication between both engineered E. coli and V. harveyi.

Materials and methods

Strain construction can be found here ( Protein production quorum sensing molecule extraction NMR analysis Cultivation conditions medium composition

Results and discussion

The figure 1 presents the results obtained with the supernatants of wild type E. coli (as a negative control with no C8-CAI-1 produced), wild type V. harveyi (as a positive control producing C8-CAI-1), and our engineered E. coli strain expressing cqsA from V. harveyi (i.e. VhCqsA, Part:BBa_K2278001 . These supernatants were analysed by ¹H Nuclear Magnetic Resonance (NMR). Characteristic and observable proton signals of C8-CAI-1 in ¹H NMR should appears at approximatively (δ, in ppm) 4.14 as a triplet dedoubled (CH in α of both carbonyl and hydroxyl groups), 2.50-2.36 (CH2 in α of carbonyl group) and 1.92-1.84 (1H of CH2 in β of hydroxyl group) as multiplets. Unfortunately, the NMR profiles of the three supernatants were similar and no characteristic ¹H NMR signal of C8-CAI-1 had been detected neither in wild type V. harveyi nor in E. coli-VhCqsA strains. So, NMR approaches failed to confirm the production of C8-CAI-1 molecule in both E. coli-VhCqsA and in V. harveyi WT strains. This does not mean the tested construction is not functional but more probably that C8-CAI-1 production was below the sensitivity limit of NMR. As a more sensitive alternative, mass spectrometry analyses were also applied on the same samples. However, no conclusive results were obtained (data not shown) and to the optimization of the method required extra time. We thus cannot exclude that the expression of CqsA is not functional but a more sensitive approach such as in vivo strategy should be tested to check the functionality of this part.

Fig. 1: Detection of C8-CAI-1 by NMR analysis (500MHz, CDCl3, 298K) in culture supernatant. Overlaid ¹H NMR spectra of freeze-dried supernatants from E. coli-pSB1C3 (negative control) in green, E. coli-VhCqsA (assay) in red, wild type V. harveyi (positive control) in blue. Supernatant have been freeze-dried and re-suspended into deuterated chloroform (CDCl3) before ¹H NMR analysis.

Perspectives

Culture parameters, induction by IPTG or extraction protocol could be optimized to increase C8-CAI-1 production. Alternatively, MS method could be improved to detect the C8-CAI-1. Alternatively, we can focus on the expression of the cqsA gene from V. cholerae to create a safe V. cholerae effector.

2. E. coli producing C8-CAI-1 molecules can be sensed by V. harveyi

Background

In response to its quorum sensing molecule, i.e. C8-CAI-1, V. harveyi becomes bioluminescent. Here we used it as a sensor to detect the production of C8-CAI-1 by our engineered E. coli-VhCqsA strain (i.e., Part:BBa_K2278001 .

Materials and methods

Protocols can be found here: Medium composition , Solid bioluminescence assay

Results and discussion

We used the JMH626 mutant strain of V. harveyi, a strain with deletion of all its quorum sensing production genes, including cqsA^{1. Thus, it is unable to produce its own C8-CAI-1. However, the whole apparatus for response to quorum sensing is still present in this strain. So, the supernatant of the E. coli-VhCqsA strain was applied on this strain to check if it was able to trigger luminescence, as a natural property of V. harveyi exposed to C8-CAI-1. Results are presented in Figure 2. In the negative control with V. harveyi ∆cqsA supernatant applied to the same strain, a very low basal bioluminescent is observed (likely from promoter leaking). The same very low basal luminescence level was observed with supernatant of wild type E. coli strain. As expected, when the supernatant of V. harveyi wild type strain was applied to V. harveyi ∆cqsA, bioluminescence could be restored. When supernatant of E. coli-VhCqsA strain was applied to the V. harveyi ∆cqsA strain, bioluminescence was observed with a comparable level to the one observed with wild type V. harveyi. Together, these data demonstrate that C8-CAI-1 is efficiently produced by E. coli-VhCqsA and thus validate our BBa_K2278001 part. More importantly, this result also demonstrates that we successfully created synthetic communication between engineered E. coli and V. harveyi strains.}

Fig. 2: Solid bioluminescence assay. Two strain of V. harveyi were plated (WT and ΔcqsA) with the addition of supernatant from V. harveyi WT, V. harveyi ΔcqsA and supernatant from E. coli with an empty pSB1C3 vector (E. coli) or E. coli with pSB1C3-cqsA (E. coli-cqsA/i>). This picture is a representative picture of the 4 times repeated experiment.

Perspectives

Next step will be to proof the communication between E coli producing the CAI-1 from V. cholerae ( Part:BBa_K2278002 ) and V. harveyi expressing the gene encoding the modified sensor CqsS* that could sense both C8-CAI-1 and CAI-1.

3. Modification of V. harveyi to detect both C8-CAI-1 and CAI-1

Background

V. harveyi has all the assets to be a good sensor for V. cholerae: it possesses its own pathway of detection for C8-CAI-1, very similar to the V. cholerae pathway to detect CAI-1. It has been shown that only a single point mutation on its receptor CqsS allows V. harveyi detecting V. cholerae CAI-1 molecule^{1. Because genetic in V. harveyi is limited (i.e. classical transformation does not work), we first had to set up a triparental conjugation protocol.}

Materials and methods

Strain construction can be found ( here ) and protocols used can be found here: Triparental conjugation , Fluorescence microscopy , Cultivation conditions , Medium composition ).

Results and discussion

A protocol of triparental conjugation to transfer plasmids to V. harveyi was set up from an original conjugation method provided by M. Arlat (LIPM, Toulouse) usually used with Xanthomonas campestris. It was adapted to our V harveyi strain. It requires an E. coli donor with the conjugative plasmid pBBR1MCS-5 + insert (BBa_J04450), an E. coli helper with the helper plasmid pRK2073, and a receiver V. harveyi. Transformants were analysed by fluorescence microscopy and results are presented in Figure 3. The pictures show that the strain of V. harveyi conjugated with the plasmid pBBR1MCS-5 containing the BBa_J04450 construction is able to express RFP using a promoter and terminator from E. coli. This raised several important conclusions. (i) It clearly demonstrated that V. harveyi can be engineered. This is the first use of V. harveyi as a genetic chassis in the IGEM competition. (ii) It proved that iGEM registry elements could be used in V. harveyi (promoter and terminator for instance). (iii) To the best of our knowledge, this is the first reported use of RFP in V. harveyi.

Fig. 3: Fig. 3: V. harveyi can be modified to produce RFP. V. harveyi JMH626 (panels C & D) and conjugated with pBBR1MCS-5-RFP (panels A & B) in optic microscopy (panel A & C) or fluorescence microscopy (panels B & D). Emission: 530-560nm/Excitation 572-647nm. On the panel (A), which show control V. harveyi JMH626, no red bioluminescence is observed and the panel (C) prove the presence of cells by optic microscopy. On (B), red bioluminescence is clearly visible. The position of the red stains are correlated to with the location of the V. harveyi pBBR1MCS-5-RFP conjugated cells observed by optic microscopy (D).

Perspectives

Next step will be to integrate the plasmid containing the engineered CqsS* receptor into V. harveyi to create a sensor able to detect both V. cholerae CAI-1 and V. harveyi C8-CAI-1. Likewise, the Als construction tested in E. coli could be now tested in V. harveyi (see next module).