Team:INSA-UPS France/Description
iGEM UPS-INSA Toulouse 2017
Synthetic biology is based on Nature everlasting possibilities, usually by inserting genetic information from microorganisms into a single and unique chassis1,2,3. However, a single chassis could present inevitable limits (high genetic burden, incompatibility of some elements with the chassis, too complex design…). These start to be a limit in synthetic biology, its perspectives and applications4. An emerging solution is the use of synthetic consortium (figure 1). Synthetic consortium have the advantage to require less amount of genetic information into a single chassis to achieve the required process since 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 signaling or metabolic pathways. Several successes of those synthetic consortia, such as production of bio-electricity5 or shortening bio-manufacturing process like C-vitamin synthesis6 have provided insight into 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 feasibility 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 Escherichia coli strain mimicking Vibrio cholerae. It triggers a sensor bacterium, Vibrio harveyi, which in turn activates the effector cell (the yeast Pichia pastoris). The later eradicates Vibrio species by producing innovative antimicrobial peptides from crocodile.
While we were defining our strategy, a cholera epidemia started unfortunately to expand in Yemen7. This terrible situation led us to focus on this problematic as it appears that current solutions are not efficient enough to deal with this situation. The bacteria V. cholerae, agent of the cholera disease, is usually found in water and infects more than a million people each year.
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 on implementing the quorum sensing detection pathway of V. cholerae into E. coli to activate reporter gene expression. However these projects, no matter how clever and brilliant they might be, were not successful enough, likely due the 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. Using multiple microorganisms 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 modification to set up the required functions. The information processing steps have been split in two different microorganisms: V. harveyi as the sensor and P. pastoris as the effector, with the system triggered by V. cholerae presence.
The whole synthetic consortium is composed of three microorganisms. The first one should be V. cholerae but for safety reason, we engineered an E. coli strain to produce a V. cholerae molecular signal. The second bacteria required a quorum sensing pathway to detect V. cholerae signal. V. harveyi naturally possesses such pathway and we engineered it to make it able to sense V. cholerae. We also engineered V. harveyi to make it producing a second molecule messenger. The third microorganism, P. pastoris was engineered to detect this messenger and produce in response the secretion of a high amount of antimicrobial peptides that can lyse V. cholerae.
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. Since our sensor was V. harveyi, as a proof of concept, we also transformed an E. coli strain with the cqsA encoding gene of V. harveyi. This Vh_CqsA enzyme synthetizes C8-CAI-1, an analog of V. cholerae CAI-114.
We therefore developed E. coli strains which produce markers simulating the presence of Vibrio species in the medium.
The easiest way to detect CAI-1 or C8-CAI-1 is to use the quorum sensing pathway of the non-pathogen V. harveyi. As our project will deal directly with V. cholerae in real situation, the CqsS receptor of V. harveyi will have to also recognize the CAI-1 molecule13. To do so, a single mutation was introduced in the gene cqsS changing the phenylalanine 175 into a cysteine.
When C8-CAI-1 binds to CqsS this activate a dephosphorylation cascade leading to the inhibition of the pQRR4 promoter and blocking the transcription of siRNA14. In the absence of this siRNA, translation of the targets genes (i.e. virulence genes) is activated (See Figure 2). We used this system to produce the molecule (i.e. diactetyl) used to activate P. pastoris . The als coding sequence, encoding for the acetolactate synthase Als, is involved in the conversion of endogenous pyruvate into diacetyl (Figure 3). als was placed under the control of pQRR4 promoter. In this engineered V. Harveyi strain , diacetyl production will be produced in response to both CAI-1 or C8-CAI-1.
The function of the third partner is to efficiently produce a killing molecule (i.e. anti-microbial peptides, AMPs) to lyse both V. cholerae and V. harveyi. This microorganism has to be resistant to the AMPs that are specific to prokaryotic cells. Therefore we chose an eukaryotic cell. Last this eukaryotic microorganism has to communicate with prokaryotic cell. Team SCUT 15 previously described a binding-receptor system involving diacetyl and a 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 through the endogeneous Ste12 pathway (Figure 4). For all this reasons, we chose P. pastoris as the effector organism since it already possess the ste12 pathway and is good protein producer 18,19.
We engineered the yeast to secret the AMPs under control of the pFUS1 promotor. We choose the AMPs from crocrodiles 20,21,22,23. Indeed crocodiles display a remarkable and efficient defense system, allowing the reptiles to resist to a large spectrum of bacterial 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.
See our Design page for more informations about the genetic elements we used!
Description
Synthetic biology: from unique chassis to synthetic consortia
Genesis of our molecular strategy
A microbial consortium chassis against cholera
Mimicking Vibrio cholerae using Escherichia coli
The sensing organism: Vibrio harveyi
The effector 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
Strategy pages
Description
Design
Parts
