Synthetic biology: to the multi-organisms communication and beyond

Nature is still developing a wide large diversity of remarkably efficient pathways in order to sense presence of specific chemical, or even physical parameters such as temperature, pressure and light^1,2. While biology originally described these phenomena, synthetic biology emerged to take advantage of Nature’s tricks, basically by inserting genetic information from microorganisms into a single and unique one, most of the time Escherichia coli³. However, focusing only on this type of bacteria 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.

Then, our iGEM project focused on a multi organisms communication pathway, especially between prokaryotic and eukaryotic cells. Thus, we developed a strategy using a cascade of events from a sensor cell (Vibrio harveyi) to an effector cell (Pichia pastoris) in order to detect and eradicate a Vibrio cholera mimicking cell (Escherichia coli) using an antimicrobial peptides from crocodile.

Genesis of our molecular strategy

During our iGEM brainstorming, while defining our strategy, cholera epidemic started unfortunately to expand in Yemen⁵. Actually, the bacteria Vibrio 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 Vibrio cholerae^6,7. Additionally, some iGEM teams tried also to deal with the challenging detection of V. cholerae^8,9,10, using E. coli. They based their strategy around the quorum sensing system of V. cholerae in order to detect it, implementing CqsS receptor and the LuxU/O pathway into E. coli in order 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. That is why we built a synthetic consortium of microorganism against Vibrio cholerae.

A microbial consortium chassis against cholera

We finally created an artificial consortium chassis to deal with cholera disease. The different partners are described below.

• To mimic V. cholerae by producing CAI-1 molecule. This will be done in E. coli
• A bacteria with a quorum sensing pathway activated on the V. cholerae presence on which CAI-1 bind. Vibrio harveyi naturally possess that pathway. It will lead to the production of a messenger molecule: that we choose to be diacetyl.
• The diacetyl binds to the Odr-10 receptor that can be expressed on yeast such as Pichia pastoris and start a molecular pathway.This pathway lead to the activation of pFUS1 and will produce our antimicrobial peptide with a secretion cassette. Those peptides will kill Vibrio cholerae.

Mimicking Vibrio cholerae using Escherichia coli

An interesting property of Vibrio cholerae is its quorum sensing autoinducer system based on the production of CAI-1 molecule¹¹. The amount of this secreted molecule, produced by the enzyme CqsA synthase, is a good reporter of the quantity of bacteria in water. As we were not allowed to work with pathogens in our lab, we engineered the strain Escherichia coli in order to mimic V. cholerae. Thus, we transformed E. coli strain with the CqsA synthase coding gene of Vibrio harveyi, non-pathogen bacteria. CqsA from V. harveyi produces an analog of CAI-1, the molecule C8-CAI-1, from (S)-adenosylmethionine (SAM) and octanoyl-coenzyme A12. 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.

The sensing organism: Vibrio harveyi

Once we developed E. coli to produce the V. harveyi 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, CqsS¹². For example, pQRR4 is a promoter which activation depends on the presence of C8-CAI-1. To fit with CAI-1 molecule, the CqsS receptor of V. harveyi only needed to be mutated on a single amino acid. We only had to mutate CqsS changing the phenylalanine 175 into a cystein and to integrate the ALS gene under the control of pQRR4 to trigger diacetyl production in presence of CAI-1¹².

We checked the metabolism of diacetyl of V. harveyi on KEGG Pathway, and identified that the acetolactate synthase (ALS) alone allowed the production of diacetyl from pyruvate, a ubiquitous metabolite.This is the second step of our molecular cascade.

The effecting organism: Pichia pastoris

The molecular response to the presence of the mimicking vibrio E. coli strain is the production by V. harveyi of diacetyl. We then need a third partner to produce toxic molecule to kill V. cholerae. This last partner needs to be resistant to the toxic molecule so we choose an eukaryotic cell. Team SCUT¹³ previously described a binding-receptor system involving diacetyl and an eukaryotic receptor, the Odr-10 receptor^14,15. 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. Pichia pastoris has been chosen as it displays already the Odr-10/pFUS1 pathway.

Moreover, P. pastoris is a good protein producing organism^16,17. We engineered the yeast to secret the toxic molecule under the promoter of Ste12. The toxic molecule secreted by P. pastoris is originated from crocodiles^18,19,20,21. Crocodiles display a remarkable and efficient immune system, allowing the reptiles to resist to a large spectrum of diseases. 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 death²². This is the third step of our cascade.

Our system

See our Design page for more informations of the genetic engineering we used!

References

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