Team:INSA-UPS France/Description
iGEM UPS-INSA Toulouse 2017
Nature is still developing a large diversity of remarkably efficient pathways in order to sense presence of specific chemical, or even physical parameters such as temperature, pressure and light1,2. While biology originally described these phenomena, synthetic biology emerged to take advantage of Nature’s tricks, usually by inserting genetic information from microorganisms into a single and unique chassis3. Focusing only on this type one microorganism is not appropriate to reflect the large complexity of living organisms and more, their intimate relationship in Nature. This aspect starts to be a limiting border in the way of the development of the synthetic biology4. An emerging solution is the use of synthetic consortium (figure ). Synthetic consortium have the advantage to require less amount of genetic information into a single chassis to achieve the required process: different microorganisms can share the genetic burden. Moreover, the components of the consortium could be selected to reduce the genetic modifications and increase the chance of success, for example, by taking advantage of already existing signalling or metabolic pathways into more exotic organisms to drive a desired process look more appealing than a full integration of those pathways on a well known genetic chassis such as E.coli. Several successes of those synthetic consortia, such as generating bio-electricity5 or shortening bio-manufacturing process like C vitamin production6 have provided insight of the strength of this approach.
Such approaches are still rare in the iGEM competition, maybe because they require to combine classic strain engineering with information processing strategies. So, our challenge was to demonstrate the power and faisability of synthetic consortium approach to open new perspectives and applications to iGEMers.
As a proof of concept, we developed a strategy against cholera. It is based on a cascade of events starting from an engineered E. coli strain mimicking Vibrio cholerae. It triggers a sensor bacterium, Vibrio harveyi, that in turn activates the effector cell (the yeast Pichia pastoris). The later eradicates V. cholerae by producing innovative antimicrobial peptides from crocodile.
During our iGEM brainstorming, while defining our strategy, cholera epidemic started unfortunately to expand in Yemen7. Actually, the bacteria V. cholerae that causes cholera disease is usually found in water and infects more than a million of people each year. This terrible situation led us to focus on this problematic and it appeared that current solutions were not efficient enough to deal with this situation.
Recently, academic research groups started to focus on synthetic biology in order to find a way to deal with V. cholerae8,9. Additionally, some iGEM teams tried also to deal with the challenging detection of V. cholerae10,11,12, using E. coli. They based their strategy around the quorum sensing system of V. cholerae in order to detect the bacteria, implementing CqsS receptor and the LuxU/O pathway into E. coli to activate gene expression. However these projects, no matter how clever and brilliant they might be, were not successful enough maybe due to the process complexity of introducing a large amount of DNA information in a single microorganism. This is the reason why we built a synthetic consortium of microorganisms. Each step of the cell to cell communication has been split in three different microorganisms. Using multiple microorganism instead of one will allow us to choose existing species that are already specialized for their tasks, as well as reducing the amount of necessary DNA to gain novel functions. Those microorganisms are V. cholerae, V. harveyi and P.pastoris and the consortium will start acting upon the quantity of V. cholera and its action will end by the death of the initiator.
This synthetic consortium is composed of several microorganisms. The first one would be V. cholerae but for safety reason we engineered an E. coli strain to produce a V. cholerae molecule marker. The second bacteria displays a quorum sensing pathway, as a central point of the cell to cell communication. V. harveyi naturally possesses this molecular pathway and we dedicated this quorum sensing to the detection of the presence of the V. cholerae mimicking E. coli and to produce a second molecule messenger. The third microorganism detects this messenger and induces the expression of a high amount of antimicrobial peptides to lyse V. cholerae. P. pastoris fulfills these requirement as the pFUS1 activation mediated by the diacetyl receptor ODR10 can be functionally expressed in yeasts.
We finally created an artificial consortium chassis to deal with cholera disease. The different partners are deeper described below.
An interesting property of V. cholerae is its quorum sensing autoinducer system based on the production of CAI-1 molecule13. The amount of this secreted molecule, produced by the enzyme CqsA synthase, is an efficient reporter of the quantity of bacteria in water. As we were not allowed to work with pathogens in our lab, we engineered the strain E. coli in order to mimic V. cholerae. E. coli was transformed with the cqsa gene from V. cholerae but as a proof of concept, we also transformed E. coli with the cqsa coding gene of V. harveyi, a non-pathogen bacteria. The enzyme CqsA from V. harveyi synthetizes an analog of CAI-1, C8-CAI-1, from (S)-adenosylmethionine (SAM) and octanoyl-coenzyme A14.
Finally, we developed an E. coli strain which produces a marker simulating the presence of the pathogen V. cholerae in the medium. This is the first step of our molecular cascade.
Once we developed E. coli to produce the C8-CAI-1, this molecule as to be detected in the medium. The easiest way to do it is to use directly the quorum sensing of the non-pathogen V. harveyi. This bacteria is an advantageous good engineerable chassis. We identified that V. harveyi already possesses gene expression depending on the binding of C8-CAI-1 on its receptor, CqsS14. Thus, pQRR4 is a promoter whom activation is C8-CAI-1 dependent.
When our project will deal directly with V. cholerae in real situation, the CqsS receptor of V.harveyi will have also to recognize the CAI-1 molecule 13. To do so, a single mutation was introduced in the gene cqss changing the phenylalanine 175 into a cysteine. As a consequence, V. harveyi is able to induce expression of a gene under the control of the promotor pQRR in response to both CAI-1 and C8-CAI-1. The gene we placed under the control of pQRR is als, coding for the acetolactate synthase ALS. This enzyme is involved in the conversion of endogenous pyruvate into diacetyl 15(Figure 3).
This is the second step of our molecular cascade, inducing a molecular response of V. harveyi to the presence of the mimicking vibrio E. coli strain through the production of diacetyl.
Previous bacteria are thus involved in the detection of V. cholerae. The response is the production of diacetyl. Then, a third partner is required to produce toxic molecule to lyse V. cholerae. Furthermore, this microorganism has to be resistant to the toxic molecule itself, and so we choose an eukaryotic cell. Team SCUT 15 previously described a binding-receptor system involving diacetyl and an eukaryotic receptor, Odr-10 16,17. It is a G Protein Coupled Receptor isolated from Caenorhabditis elegans that once activated by diacetyl, lead to the activation of the pFUS1 promoter by Ste12. P. pastoris is then the perfect candidate, as it displays already the Odr-10/pFUS1 pathway and is also a good protein producer 18,19.
We engineered the yeast to secret the toxic molecule under the promoter of Ste12. The toxic molecule secreted by P. pastoris is originated from crocodile 20,21,22,23. Crocodiles display a remarkable and efficient immune system, allowing the reptiles to resist to a large spectrum of bacteria infection. Thus, they produced antimicrobial peptides (AMPs) which are able to lyse bacteria such as V. cholerae. AMPs are cationic pore-forming molecules targeting bacterium membranes, causing bacterial lysis and death 24.
This is the third step of our cell to cell communication pathway.
See our Design page for more informations of the genetic engineering we used!
Description
Synthetic biology: to the multi-organisms communication and beyond
Genesis of our molecular strategy
A microbial consortium chassis against cholera
Mimicking Vibrio cholerae using Escherichia coli
The sensing organism: Vibrio harveyi
The effecting organism: Pichia pastoris
Our system
References
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246677/
https://www.ncbi.nlm.nih.gov/pubmed/26308982
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4531478/
https://www.ncbi.nlm.nih.gov/pubmed/16697733
https://www.ncbi.nlm.nih.gov/pubmed/22001326
https://www.ncbi.nlm.nih.gov/pubmed/21219472
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC23737/
https://www.ncbi.nlm.nih.gov/pubmed/2302121
https://www.ncbi.nlm.nih.gov/pubmed/27905091
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494115/
https://www.ncbi.nlm.nih.gov/pubmed/21184776
https://www.ncbi.nlm.nih.gov/pubmed/2059789
https://www.ncbi.nlm.nih.gov/pubmed/28159460
https://www.ncbi.nlm.nih.gov/pubmed/24192554
https://www.ncbi.nlm.nih.gov/pubmed/27837316
