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Team:UNITS Trieste/Project
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Revision as of 22:34, 5 September 2011
SYNBIOME OVERVIEW
An important challenge in the near future will be the optimization of bioreactors for the production of complex molecules. The aim of our research project is to combine different cell systems commonly used in biosynthesis through synthetic biology. To improve this system we want to use cells from different kingdoms because we believe that different cell types could cooperate and better produce complex molecules. The innovation and challenge will be to obtain a stable community of cells from different kingdoms and establish mutualism among them. This interdependence will be obtained through a metabolic and signaling pathways in which the survival and/or growth depends from the other cell types.
The project is based on a three-element system: two different bacterial strains and one eukaryotic cell type that communicate through quorum sensing (QS) signal molecules.
In order to achieve the goal in constructing this synthetic community, both the bacterial cells and the eukaryotic cell will be engineered with a genetic circuit under the regulation of the N-acyl homoserine lactone (AHL) QS signals.
More specifically, we will engineer both bacterial strains to produce the enzyme cellobiosidase, in order to convert extracellular cellobiose into glucose, while the eukaryotic cell will be engineered to produce a soluble form of beta-lactamase.
This set up will ensure interdependence among the three cell types; all cells will benefit from the free available glucose and the two bacteria will survive in an ampicillin-containing culture medium. The mutalism between the two different bacterial strains will occur thanks to a synthetic network based on the two different AHL QS signals, namely 3-oxo-C8-AHL and 3-oxo-C12-AHL.
The inter-kingdom mutualism will be guaranteed by an eukaryotic trans-activator sensible to the AHL QS mediator 3-oxo-C8-AHL.
Importantly, this genetic circuit will be designed in such a way so that it can be adapted to different bacterial species and eukaryotic cell types.
pTraBox
P65-TraR
AHL Sensible Eukaryotic Switch
We decided to test both pTraBox-SEAP and p65-TraR (Neddermann P. et al., 2003), kindly provided by Dr. R. Cortese's group, using SEAP (Secreted alkaline phosphatase) as reporter gene, detected with the Great Escape Chemiluminescent assay kit (Clontech).
In our final system we aim to have the presence of both the OXOC8 and the OXOC12 but the eukaryotic cell has to be sensible only to OXOC8.
On this basis the assay was performed in order to test the efficiency of this inducible promoter after the induction with OXOC8 and the response to OXOC12 as unspecific ligand.
AHL has to be dissolved in a organic solvent as Ethyl-Acetate in order to prevent the lactonolysis that will occur in prolonged exposure to aqueous conditions.
2x105 cells for Hela were placed in 35mm culture dishes and transfected using the Fugene HD transfection reagent (Promega). For each transfection 2ug of DNA were transfected.
For all the experimental conditions that we tested, were performed biological triplicates and experimental triplicates.
Figure 1. SEAP activity12 hours after transfection.
2x105 cells for Hela were placed in 35mm culture dishes and transfected using the Fugene HD transfection reagent (Promega). For each transfection 1ug of transactivator plasmid (P65-TraR) and 1ug of pTraR-SEAP reporter were transfected. We decided also to test the basal activity of SEAP under the control of TraBox-CMVmin, in order to achieve this goal hela cells were transfected with 1ug of pTraR-SEAP and 1ug of pCDNA3.
After 6 hours 20uM of AHLs (OXOC8 and OXOC12 separately) were added to cell culture medium and 12 hours after the addition of ligands the medium was collected and the activity of SEAP was measured.
Hela WT were treated with a corresponding amount of Ethyl Acetate + OXOC8.
Figure 2. SEAP activity 24 hours after transfection.
2x105 cells for Hela were placed in 35mm culture dishes and transfected using the Fugene HD transfection reagent (Promega). For each transfection 1ug of transactivator plasmid (P65-TraR) and 1ug of pTraR-SEAP reporter were transfected. We decided also to test the basal activity of SEAP under the control of TraBox-CMVmin, in order to achieve this goal hela cells were transfected with 1ug of pTraR-SEAP and 1ug of pCDNA3.
After 6 hours 20uM of AHLs (OXOC8 and OXOC12 separately) were added to cell culture medium and 24 hours after the addition of ligands the medium was collected and the activity of SEAP was measured.
Hela WT were treated with a corresponding amount of Ethyl Acetate + OXOC8.
Figure 3. Luciferase activity12 hours after transfection.
2x105 cells for Hela were placed in 35mm culture dishes and transfected using the Fugene HD transfection reagent (Promega). For each transfection 1ug of Luciferase reporter plasmid and 1ug of pCDNA3 reporter were transfected as positive control of trasnfection. Cells were treated with 20 uM OXOC8 6 hours after trasnfection.
