Team:Pasteur Paris/Demonstrate

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NERVE GROWTH FACTOR AND NEURONAL CULTURE

We successfully designed and cloned a biobrick coding for the secretion of rat proNGF in pET43.1a and iGEM plasmid backbone pSB1C3, creating the new part Bba_K2616000 . We confirmed this genetic construct by sequencing.


We confirmed the production of proNGF in three different ways:

  • SDS PAGE followed by western blot.
  • Mass spectrometry.
  • Activity on the growth of rat E18 cortical cells.

We designed and manufactured microfluidic chips in order to test our final proof of concept.

We grew embryonic E18 rat neurons in our self-made microfluidic chips and successfully observed axon growth in the presence of commercial NGF and our recombinant proNGF.

We showed that the activity of our recombinant proNGF was comparable to the one of commercial NGF used at concentrations of 500 to 900 ng/mL.

KILL SWITCH

We successfully designed and cloned a biobrick coding for a temperature sensitive kill-switch, creating the new part Bba_K2616002 . We confirmed this genetic construct by sequencing.


We observed the survival and normal growth of our engineered chassis E. coli BL21(DE3)pLysS at 25°C and 37°C and the absence of growth at 18°C and 20°C. Comparatively, our negative control, the same chassis, transformed with an empty vector, grew normally at all temperatures tested.


To conclude, we have demonstrated that our kill-switch is a very efficient way to prevent the contamination of the environment by our genetically modified bacteria in case of accidental release.

MEMBRANE BIOCOMPATIBILITY AND CONDUCTIVITY

In search of a biocompatible conductive polymer to confine bacteria, we successfully polymerized PEDOT:Ts and PEDOT:Cl on alumina oxyde membrane filters. We also partially polymerized PEDOT:PSS.

We demonstrated that a polymer-coating enhances the electrical properties of the membranes as PEDOT:Ts-coated and PEDOT:Cl-coated membranes are more conductive than uncoated membranes. Moreover, experiments showed a better biocompatibility for the polymer-coated membranes compared to the gold-coated ones.

We successfully demonstrated that our membrane could confine bacteria. This feature is one of the essential safety components that we have engineered in our NeuronArch project, ensuring the patient's safety.

DESIGN