Team:Pasteur Paris/Demonstrate
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 a 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 a new part Bba_K2616002 . We confirmed this genetic construct by sequencing.
We observed survival and normal growth of our engineered bacteria at 25°C and 37°C and absence of growth at 18°C and 20°C, showing the efficiency of the kill switch if our bacteria are released in the environment.
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 properties of the membranes as PEDOT:Ts-coated and PEDOT:Cl-coated membranes are more conductive than other membranes. Moreover, experiments showed a better biocompatibility for the polymer-coated membranes compared to the gold-coated ones.
We succeeded in finding appropriate membranes for our project, namely PEDOT:Ts and PEDOT:Cl. We would like to do further research and improve the way we polymerized PEDOT:PSS because it is widely used in organic electronic.