Proof Of Concept
Step 1 : Initial idea from literature
The first step for our proof of concept was to build knowledge from literature and previous experiments. For phase I, the use of a cobalamin riboswitch came mainly from previous iGEM teams (Paris Bettencourt 2015, OUC China 2019). For phase II, the idea for the development of a bioelectronic system using Shewanella oneidensis came from a previous Ionis Paris iGEM team (Cinergy, 2019), and mainly from the work of Zeng et al. in 2019 .
Step 2 : Theoretical Implementation
Following the theoretical building of our project, different parameters for each Phase were set up (See Description). Implementation parameters were set up on imaginating and doing some research on potential users, evaluating risks and challenges, and targeting the appropriate regulation. This step set up specific specifications for our laboratory and human practice works. For more information on this step, see Implementation.
Step 3 : Human Practices
With information gathered from steps 1 and 2, we built the bases of our project. Human practices work allowed us to strengthen the scope of our project by discussing with different actors of our project’s environment (Consumers, organizations, doctors and specialists, regulators and public). Thanks to these discussions we truly understood the potential of our project and the reel need for such a system, even beyond vitamin B12 detection, by combining our bioelectronic prototype with previous iGEM projects. For more information on this step, see Human Practices.
Step 4 : Development of a prototype and lab work
By gathering all the other aspects of our projects, we were able to structure our lab investigations around two main phases :
Phase 1 : the production of lactate under stimulation of vitamin B12
As explained in results part, we did not manage to assemble our biobricks in order to prove that vitamin B12 concentrations could be reported with the production of lactate, using the cobalamin riboswitch to express lactate dehydrogenase.
However, results from OUC China 2019 suggest that the cobalamin riboswitch could be used as an efficient structure for the reporting of low vitamin B12 concentrations. Moreover, this structure appears to be sensitive to vitamin B12 low concentrations.
Figure from OUC China 2019 : “The response curve of our improved cobalamin biosensor to Vb12. As we expected, an obvious decrease of mRFP1 expression was observed with the increased concentration of vb12. It demonstrates that the modular riboswitch functions properly.”
For the second part of phase 1, results from Zeng et al. in 2019 suggest that metabolite production using an enzyme is an efficient way of reporting gene expression .
Phase 2 : Development of a bioelectronic sensor of lactate
As explained in results, we were able to build a simple lab-scale protocol for the recording of Shewanella oneidensis electrical activity under metabolic stimulation. This prototype was thought to be easily reproduced in the laboratory :
By using this prototype, the following measures were taken under lactate stimulation of Shewanella oneidensis :
When taking the control as a blank, the following curve is obtained :
These results suggest that lactate concentrations can be reported by Shewanella oneidensis in a few hours with our prototype. Of course, more experiments have to be performed to confirm this tendency.
Following the set up of this lab-scale prototype, we modelled and printed a portable prototype. Hardware and software work allowed us to automate the process of voltage recording for smaller-scale recordings :
3D-printed prototype for the recording of Shewanella oneidensis activity
Our final Proof Of Concept cycle can be sumarized as followed :
Cobatect’s proof of concept final cycle (created with Biorender)
 Zeng, J., Banerjee, A., Kim, J. et al. A Novel Bioelectronic Reporter System in Living Cells Tested with a Synthetic Biological Comparator. Sci Rep 9, 7275 (2019). https://doi.org/10.1038/s41598-019-43771-w