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.
To conclude, 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, demonstrating that our theoretical idea works in vitro.
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.
To conclude, 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.
We succeeded in conceptualizing an ergonomic and functional amputated stump-to-prosthesis interface device and charging station, at the scale of a prototype. We thought about its future design at an industrial scale. We also succeeded in modeling in 3D the entirety of the components of our device and of its charging station. We integrated a pragmatic and rapid quarter turn system for the osseointegrated stem so that patients are capable of putting on and taking off the device with ease.
We printed scale models using stereolithographic techniques. The thickness of the device's shell was first too substantial, which caused polymerization problems and tension between the two shells. We took into account those results, reduced the thickness and 3D-printed again the models, gaining in precision and lightness (-25% of the total weight).
The electronic schematics for the induction charging system, as well as the LED loop, allowing the visualization of charging and synchronizing of the device was also added to the device. The electronic parts are perfectly fitted into the 3D-printed device, and the charging system is totally operational.
We conceived a usage scenario, as well as a smartphone application.
To conclude, we tried to think of all the aspects of our device, from security, to manufacture, and ergonomy. We demonstrated a complete approach of product design incorporating a synthetic biology solution to nerve growth and biofilm infections in prostheses.
We successfully realized a study target market analysis, allowing us to visualize the population NeuronArch was aiming at and its evolution over the coming years.
We studied and analyzed our possible competitors, such as Ossur, Integrum, Ottobock, etc. and analyzed our advantages compared to them. We designed a marketing plan, showed our sales plan and communication strategy. We also talked about our current (and possible future) sponsors and collaborators, as well as the crowdfunding.
We also showed how we managed our budget during this iGEM competition to bring all 19 team members and 4 coaches to Boston in addition to our lab expenses, and how we plan to budget in the future.