Team:INSA-UPS France/Results
iGEM UPS-INSA Toulouse 2017
The experimental results described here provide insights of the functionality of our project and clues about how our synthetic consortium will work.
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. cholerae 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.
Strain construction can be found here ( clonings ) and protocols used can be found here Protein production and sampling, NMR analysis , Cultivation condition and Medium composition ).
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.
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.
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 )
.
Protocols can be found here: Medium composition , Solid Bioluminescence assay.
We used the JMH626 mutant strain of V. harveyi, a strain with deletion of all its quorum sensing production genes, including cqsA1. 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.
Next step will be to prove the communication between E coli producing V. cholerae CAI-1 ( Part:BBa_K2278002 ) and V. harveyi expressing the gene encoding the modified sensor CqsS* that could sense both C8-CAI-1 and CAI-1 (see next Results module).
V. harveyi has many 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 molecule1. Because genetic in V. harveyi is limited (i.e. classical transformation does not work), we first had to set up a triparental conjugation protocol.
Strain construction can be found here and protocols used can be found there: Triparental conjugation , Fluorescence microscopy , Cultivation condition , Medium composition.
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-4 + insert (BBa_J04450), an E. coli helper with the helper plasmid pRK2073, and a receiver V. harveyi.
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).
The quorum sensing signal detected by V. harveyi has to be transmitted to the yeast P. pastoris. For that, we chose to engineer V. harveyi so that it will conditionally produce diacetyl as the communication molecule between the bacterium and the yeast. Here, we tested the functionality of our construction containing the als gene, encoding the acetolactate synthase that enable diacetyl production ( Part:BBa_K2278011 ).
Strain construction can be found here and protocols used can be found there: Cultivation condition, NMR analysis and Protein production and sampling.
To check the functionality of the plasmid expressing als, we first used E. coli. Figure 4 presents the results obtained for the 1H NMR analysis of the supernatant of E. coli strains with empty pSB1C3 or pSB1C3-als. Diacetyl specific shift was confirmed by spiking experiment. A small peak of diacetyl could be detected in the strain expressing als but not in the control. However, the amount seems very low. This could be related to two punctual mutations observed in the als sequence of this clone. However, only a single sequencing run was performed for this part, which is insufficient to be affirmative about the reality of these mutations.
Next step will be to verify the sequence of als and to express the plasmid encoding for the right sequence of Als in V. harveyi. Diacetyl production will be then checked again by NMR.
To allow a communication between V. harveyi and P. pastoris mediated by diacetyl, the Odr-10 receptor (design) had to be expressed in P. pastoris. To check if Odr-10 was functional in vivo, we used a pFUS1-RFP reporter system. Indeed when diacetyl binds to Odr-10 a cascade of activation of Ste proteins (endogenous to P. pastoris) will lead to the binding of Ste12 on pFUS1 promoter, and the expression of RFP should be activated.
Strain construction can be found here and protocols used can be found there: Cultivation condition , Medium composition and Plate reader.
We tested the functionality of the complete system Odr-10/pFUS1-RFP by growing the cells on a medium specifically designed to induce the activation of Ste proteins (Johnston et al., 1977). Diacetyl was added at various concentration. Absorbance and fluorescence production by P. pastoris strain having integrated the empty plasmid or the plasmid containing Odr-10/pFUS1-RFP system was followed over the time on a microplate reader. A positive control was obtained by growing a Pichia strain with a constitutive high expression of RFP (pGAP promoter fused to RFP gene). Results are presented in Figure 5.
Next step will be to grow together our V. harveyi expressing Als and our P. pastoris expressing Odr-10/pFUS-RFP to demonstrate synthetic communication between them.
The final goal of our synthetic consortia was to kill V. cholerae with newly described antimicrobial peptides from crocodile. Here, we tested the production by P. pastoris of three inovative antimicrobial peptides (AMP), i.e. D-NY15, cOT2 and Leucrocin I (respectively Part:BBa_K2278021, Part:BBa_K2278023 and Part:BBa_K2278022 )
Strain construction can be found here and protocols used can be found there: Cultivation condition , Medium composition , Plate reader, Semi quantitative RT-PCR and On plate toxicity assay.
Because our AMPs have never been produced by P. pastoris and their toxicity on this yeast was not assessed, we first validated the expression of the AMPs by RT-qPCR. Figure 6 showed the results obtained for D-NY15 expression in P. pastoris. The amount of fluorescence provided by the RT-qPCR with the D-NY15 amorces (curves A1, A2 and A3) is raising after few cycles (8.32 +/- 0.03) whereas the negative control (pPICZα only, curves C1, C2 and C3) starts to be amplified at over 29 cycles (i.e. non-specific amplification). This means that the D-NY15 encoding gene is expressed in P. pastoris and non-lethal.
To test the toxicity of the three AMPs, disks soaked with the supernatant of P. Pastoris with genomic integration of pPICZα, pPICZα-D-NY15, pPICZα-cOT2 or pPICZα-Leucrocin I were put on freshly inoculated solid culture of V. harveyi. Results are presented on Figure 7. Inhibition haloes were observed around both the positive control and the patch containing the supernatant from a D-NY15 expressing strain. No growth inhibition were observed around the disks containing the supernatant from COT2 and Leucrocin I expressing strains. These data nicely demonstrated the capacity of the engineered crocodile peptide D-NY15 to inhibit V. harveyi growth but also the capacity of P. pastoris to produce antimicrobial peptides. This is the first demonstration of AMP production by P. pastoris by an IGEM Team and the first use of sequence coming from crocodile in iGEM!
Next step will be to prove the inhibitory activity of AMPs in liquid medium containing both modified P. pastoris with V. harveyi. For cOT2 or Leucrocin I, which cytoxicity was not demonstrated yet, a higher concentration of supernatant could be tested.
Ultimately our synthetic consortia will be freeze-dried and placed into a plastic bag containing dried cultivation medium. As each microorganism of the consortia has its own cultivation requirements, a difficulty was to find a common media on which V. harveyi and P. pastoris can grow together.
Protocols used can be found here: Cultivation condition , Medium composition.
P. pastoris and V. harveyi were grown on six media corresponding to variants of their standard media (LB, LB acetate 2%, LM, LM acetate 2%, YPD, and YPA 1%, see Medium composition for details).
As expected, V. harveyi and P. pastoris were growing well on their respective usual media (LM and YPD ; figure 8), but not as performant on each other medium. Acetate inhibited growth of both microorganism in YPA & LMA but it was better tolerated in LBA. Eventually, LM medium appears as good compromise to ensure a satisfying growth of both strains. This is an interesting result for this project since LM medium is cheap and easy to produce.
All these data have been used to fit our model page in order to help in the design of the device.
Next step will be to challenge the capacity of our strains to recover from freeze-dry since they will be lyophylized in our device. Preliminary tries suggested they survived but more accurate measurements will have to be performed.
We selected two types of membrane materials, i. e. TPX® X44B 50 PVDF 0.1 µm for making the sachet containing our synthetic biology system composed of V. harveyi and P. pastoris with nutrient medium. Here we tested the permeability of both to check whether this membrane could contain our synthetic organisms while allowing the antimicrobial peptides and quorum sensing molecules go through from the device compartment to the treated water.
We first used the TPX® X44B 50µm material used by iGEM Groeningen 2012 and iGEM Toulouse 2015. To assess the porosity of the material, we performed a liquid/liquid transfer assay with bengale pink dye. Bengale pink dye was chosen because of its high molecular mass for a dye (977 g/mol) to get closer to the size of our AMPs. The test was done with a Hele-Shaw measurement cell (two parallel planes spaced by 2 mm) with a camera (EDMUND OPTIC) to monitor the diffusion through the membrane (figure 9). The cell was filled with water and bengale pink was introduced from the top in a cone of membrane (or absorbent paper as a control), in contact with the cell water.
We did not observe diffusion of the dye through the TPX membrane in this experiment (Data not shown). Since we were concerned with the hydrophobicity of the material, we also tried to check if water can go through by putting the material with a drop of water on top of absorbent paper. No moisture on the absorbent paper was observed during the course of the experiment. We conclude that the chemical characteristics of the TPX material does not suit to our project.
Next step will be to test this membrane with our synthetic consortia to check whether bacterial diffusion is prevented while peptides diffusion allowed.
Results
1. Mimicking Vibrio sp. presence with an engineered E. coli
Background
Materials and methods
Results and discussion
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 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.
Perspectives
2. E. coli producing C8-CAI-1 molecules can be sensed by V. harveyi
Background
Materials and methods
Results and discussion
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.
Perspectives
3. Modification of V. harveyi to detect both C8-CAI-1 and CAI-1
Background
Materials and methods
Results and discussion
Transformants were analyzed by fluorescence microscopy and results are presented in Figure 3. The pictures show that the strain of V. harveyi conjugated with the plasmid pBBR1MCS-4 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.
Perspectives
4. Production of diacetyl to establish communication between prokaryotic and eukaryotic cells
Background
Materials and methods
Results and discussion
Perspectives
5. Diacetyl detection by Pichia pastoris:
Background
Materials and methods
Results and discussion
In the conditions without diacetyl or with 500 µM of diacetyl, no difference was observed between the control and the strain expressing Odr-10/pFUS1-RFP. However, when diacetyl is present at higher concentration (i.e. 1000 µM), higher expression is
observed than in the negative control.
These data demonstrate the functionality of the complete detection pathway. Incidently, they also show that pGAP (our positive control promoter) is functional in P. pastoris, which increase the range of application of this promoter biobrick ( Part:BBa_K431009 ). This was demonstrated again by RT-qPCR in the next Results module. Finally, this result suggests that our engineered P. pastoris is ready to communicate with Vibrio species if this former one produce diacetyl.
Perspectives
6. P. pastoris is able to produce functional antimicrobial peptides
Background
Materials and methods
Results and discussion
Perspectives
7. Co-cultivation of P. pastoris and V. harveyi is possible
Background
Materials and methods
Results and discussion
Perspectives
8. Permeability assay of the membranes used for the devices:
Background
Results and discussion
Next we tested the material proposed by our partner Sunwaterlife, i.e. a membrane used in their water cleaning system (PVDF 0.1 µm from Orelis Environnement SAS, France). We tested this material with the same Hele-Shaw measurement cell. As shown on figure 10, the dye is able to go through the material. Diffusion was observed in less than 1 minute. This means this material is entirely suitable for our project since the 0.1 µm porosity prevents bacterial diffusion but allows peptides diffusion.
Perspectives:
References:
https://www.ncbi.nlm.nih.gov/pubmed/21219472
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC221916/
Realisations pages
Overview
Notebook
Clonings
Results
Contribution
Protocols
Safety
