Team:INSA-UPS France/Design


Design

Building a synthetic consortium able to deal with the cholera issue led us to investigate on different communication pathways: we had to sense Vibrio cholerae in its natural environment and based on this sensing we had to activate, through transmission, a response: the production of a killing molecule. Moreover, all those actions had to be inserted in the right cellular chassis in order to optimize the system.

To wipe out cholera from water, we decided to build a sense-transmit-respond system reacting to V. cholerae and leading to its death.

Overview

Organisms

E. coli

We chose to mimic V. cholerae using E. coli that we modified to produce the CAI-1 of V. cholerae, using the enzyme responsible for its production. E. coli has two main advantages: it is a good molecular biology model and it produces no endogen CAI-1.

The psB1C3 plasmid was chosen for iGEM compatibility.

V. harveyi

V. harveyi has all the assets to be a good sensor for V. cholerae: it possesses its own pathway of detection of C8-CAI-1, an analogue of CAI-1. A single point mutation allows V. harveyi: to detect V. cholerae’s molecule CAI-11. Moreover, the strain that we are using, JMH626, has been deleted for the enzyme responsible of the production and detection of other quorum sensing molecules, making it a specific sensor of CAI-12. However it cannot be used as the effector because of its physiological proximity to V. cholerae. Thus we supposed that the production of antimicrobial peptides aimed at V. cholerae would be lethal to it.

The pBBR1MCS-4 broad host range plasmid was chosen so we can transfer the system into V.harveyi using a conjugation method.

P. pastoris

Recognized as a great protein producer and secretor, P. pastoris has already been used to produce a wide range of AMPs3,4. Furthermore, the diacetyl/Odr-10 system has been described as a useful tool for prokaryotic/eukaryotic communication5.

The pPICZα plasmid was chosen because of its α-factor and its homology sequence allowing it to integrate in a targeted zone in its genome. It is recognized as a good plasmid for protein production in P. pastoris.

Modules & Parts

Sense

We chose to take advantage of the intraspecies quorum sensing of V. cholerae: the CAI-1/CqsS system. To mimic this pathway in our laboratory, we had to both produce in vivo the CAI-1 molecule in a bacteria strain and express the CqsS receptor in an other one.

The CAI-1 producing system is inducible in order to avoid toxicity problems. The fact that CqsS* can detect both CAI-1 and C8-CAI-1 led us to choose V. harveyi cqsA gene (Vh-cqsA), instead of V. cholerae gene, that produces C8-CAI-1 for safety reasons.1

Transmit

V. harveyi has the natural pathway leading to the activation or inactivation of pqrr4. At high CAI-1 concentration the promoter is inactivated, thus we needed an inverter, tetR/pTet allowed us to activate the als gene that produces diacetyl at high CAI-1 concentration:

  • If CAI-1 is present in high concentration, pqrr is repressed, so TetR no longer inhibits pTet. Thus, the als gene is expressed, producing >diacetyl.
  • If there is no CAI-1, TetR is produced and repress pTet which inhibits the als gene, ergo diacetyl production.

We chose to use the diacetyl/Odr-10 binding receptor system, that is known to activate gene expression on yeasts.

The constitutive pGAP promoter allows the system to always express the Odr-10 receptor and thus be sensible to diacetyl at any time.

Respond

Once the Odr-10 receptor has sensed diacetyl, pFUS is activated and it triggers the Ste12 pathway. Then, the production of antimicrobial peptides (AMP) can start. In order for the cells to excrete the peptides, an α-factor is needed.

Experimental plan

E. coli

Quorum sensing molecule production

  • C8-CAI-1 & CAI-1 NMR
  • Bioluminescence
  • MS

V. harveyi

Conjugation

  • Conjugation test with fluorescence
  • CqsS* pathway test with fluorescence

diacetyl production

  • diacetyl NMR (E.coli and V. harveyi)
  • pTet characterization in V. harveyi by reporter gene

P. pastoris

Antimicrobial peptides (AMP)

  • AMP activity: growth tests, etc
  • AMP purification

diacetyl detection

  • pFus pathway test with fluorescence
  • etc